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The role of associated bacteria in explaining adaptation by the vine weevil Otiorhynchus sulcatus (Fabricius) to different environments by Pilar Morera Margarit A thesis submitted in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy August 2020 Candidate's declaration I, Pilar Morera Margarit, do hereby certify that this thesis has been written solely by me, and that it is the record of work carried out by me, or principally by myself in collaboration with others as acknowledged, and that it has not been submitted, in whole or part, in any previous application for a degree. Pilar Morera Margarit 2019 i Acknowledgments I firstly would like to thank my supervisors for their great support and encouragement during these four years of PhD. I would also like to thank Davide Bulgarelli, for his great support in analysing the sequencing data, and everyone in Davide´s group, especially Senga Robertson, Rodrigo Alegría Terrazas and Federica Caradonia, for their help and support in the lab. I would like to thank David Robertson for his support in designing primers, cloning and performing quantitative PCR. I would like to thank Dr Jim McNicol and Dr Katharine Preedy for their help in designing my experiments and analysing my data. I would like to thank Gill Prince for providing me with Metarhizium brunneum conidia and for helping me to learn how to grow this fungus. I would also like to thank Daniel Leybourne and Konrad Neugebauer for their help in statistics and graphics in R, I would have been lost without them. A big thanks to everyone that helped me collect vine weevils at night and also to the growers that provided us with insects. I would also like to thank Dr Dave Chandler and Dr Heather Campbell, my viva examiners, for a great discussion and for contributing to improve the content of this thesis. I would like to thank my mum Pilar Margarit Bellever, my brother Jaume Morera Margarit and my dad Jaume Morera Guitart for their support, especially my mum who has always been there in the worst and the best moments. Also especial thanks to my brother for his dedication to design fantastic figures for my papers. I would like to thank all my friends in Dundee, many of them spread now around the globe. I would also like to give special thanks to my partner Karim Hussain who, with his patience and joyful smile, has made every day better. ii Abstract The vine weevil Otiorhynchus sulcatus (Fabricius) is a curculionid pest endemic to central Europe which has successfully invaded regions on nearly all continents. This weevil species is characterised by an ability to feed on a broad range of plant species, several of which are grown as horticultural crops. Research investigating the distinctiveness of insects inhabiting separate geographic areas or infesting different crop species is still scant. Genetically, vine weevils from different locations have been shown to display limited genomic differences. The research presented here further investigated if features other than insect genetic variation are distinct amongst vine weevil populations found at different locations. The bacterial community of adult vine weevils from different populations found at geographically distinct locations within the UK but from the same host plant species was characterised. Additionally, some of these populations were tested for variation in susceptibility to a fungal pathogen commonly included in control strategies targeting this pest, Metarhizium brunneum (Petch). Oviposition choice was also examined on these insects between two host plant species susceptible to vine weevil infestation, strawberry and raspberry. Additionally, the fluctuation in titre of the bacterium Candidatus Nardonella during vine weevil development was assessed. This prokaryote was shown to dominate the bacterial community of the vine weevil and so we aimed at identifying important functions for its host. Insect population source per se did not shape the bacterial microbiota or influence the insect fitness parameters measured. Ca. Nardonella abundance followed a pattern which may indicate its involvement in tyrosine metabolism and cuticle formation. Future research should investigate vine weevils from a wider selection of geographic locations including non-crop environments to complete this line of research. Additionally, the influence of the host plant species on the distinctiveness of vine weevil populations should also be tested. Ca. Nardonella has been shown to play important roles for other weevils. Hence, the function of this bacterium for the vine weevil deserves further attention. iii Table of contents CHAPTER 1 ............................................................................................................................ 1 INTRODUCTION ....................................................................................................................... 1 1.1. Brief overview of the thesis .................................................................................... 1 1.2. The role of bacterial associations in determining the success of agriculturally important species of weevil .............................................................................................. 4 1.2.1. Tougher than tough: Candidatus Nardonella, an important player in cuticle formation ....................................................................................................................... 7 1.2.2. To fly or not to fly: Sitophilus oryzae and S. zeamais bacteria involved in flight activity .................................................................................................................. 9 1.2.3. The complicated case of weevil parthenogenesis: can Wolbachia manipulate weevil reproduction? ................................................................................................... 10 1.2.4. Back from the future: bacterial community studies in weevils, a fast-moving field 13 1.2.5. The Rosetta stone: translating bacterial community knowledge into ecological knowledge .................................................................................................. 15 1.3. A cosmopolitan and polyphagous pest: the vine weevil, Otiorhynchus sulcatus (Fabricius) ....................................................................................................................... 15 1.3.1. Catch me if you can: vine weevil control with the entomopathogenic fungus Metarhizium brunneum (Petch) ................................................................................... 16 1.3.1.1. Metarhizium brunneum pathogenesis ........................................................ 16 1.3.1.2. Metarhizium brunneum as a biopesticide to control vine weevil larvae ...... 18 1.3.1.3. Metarhizium brunneum growth is temperature dependent ......................... 19 1.3.1.4. Metarhizium brunneum in the rhizosphere ................................................. 20 1.3.1.5. Metarhizium brunneum as an endophyte ................................................... 20 1.3.2. A complete metamorphosis: the vine weevil life cycle .................................. 22 1.3.2.1. The vine weevil eggs .................................................................................. 22 1.3.2.2. The vine weevil larvae ................................................................................ 23 1.3.2.3. The vine weevil pupae ................................................................................ 24 1.3.2.4. The vine weevil adults ................................................................................ 24 1.4. Thesis aims and objectives .................................................................................. 26 CHAPTER 2 .......................................................................................................................... 27 THE BACTERIAL COMMUNITY ASSOCIATED WITH ADULT VINE WEEVIL OTIORHYNCHUS SULCATUS (FABRICIUS) IN UK POPULATIONS IS DOMINATED BY CANDIDATUS NARDONELLA ..................... 27 2.1. Introduction .......................................................................................................... 29 iv 2.2. Pilot experiment: Characterisation of the bacterial community of vine weevil adult insects from different host plant species at separate UK locations ................................ 30 2.2.1. Materials and methods ................................................................................. 30 2.2.1.1. Vine weevil adult populations ..................................................................... 30 2.2.1.2. DNA extraction ..........................................................................................