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Behavioural and olfactory responses of pest insects to endophyte- infected (Epichloë festucae variant lolii) perennial ryegrass (Lolium perenne) A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University by Louise Marie Hennessy Lincoln University 2020 Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Plant Insect Interactions. Abstract Behavioural and Olfactory Responses of Pest Insects to Endophyte-infected (Epichloë festucae variant lolii) Perennial Ryegrass (Lolium perenne) by Louise Hennessy Asexual fungal endophytes (Epichloë) colonise agricultural grasses (Poaceae) in an interaction which provides host plants with protection against herbivorous insects. Despite 40 years of research there is still much we do not know about these complex interactions. A major gap in our knowledge is an understanding of the mechanisms involved in perception of endophyte by host-searching insects. It has, perhaps, been assumed that perception of endophyte is mediated by ingestion of endophyte- derived alkaloids, resulting in a malaise and an avoidance response. Although a post-ingestional malaise is one theory it is also feasible that insects detect endophyte via sensory perception before ingesting plant material. Sensory perception involves olfactory and/or contact (gustatory) chemoreception and is often referred to as an insect’s ‘sense of smell and taste’. I explored the mechanisms involved in insect perception of the endophyte, Epichloё festucae variant lolii (Latch, M. J. Chr. & Samuels, Hypocreales: Clavicipitaceae), in perennial ryegrass (Lolium perenne Linnaeus, Poales: Poaceae) hosts. When presented with a choice a root aphid, Aploneura lentisci, was not deterred from endophyte- infected (AR37 strain) ryegrass in a host-preference assay and thus appeared to be unable to initially (24 h) perceive endophyte, demonstrating that negative effects of endophyte are not always associated with initial perception and avoidance responses. Further olfactometer experiments demonstrated that under the experimental conditions I used, apterous nymphs were unable to utilise olfaction during host-searching, suggesting that this morph cannot perceive host plants before contact is made with the plant. In contrast to this, olfaction was an important sensory mechanism for Argentine stem weevil adults (ASW, Listronotus bonariensis) which employed olfaction to locate host plants and distinguish between undamaged and herbivore damaged hosts. Olfaction is mediated by plant volatiles and both endophyte and herbivory were shown to alter the blend emitted by perennial ryegrass in this study. ii ASW perceive certain endophyte strains and I propose a role of contact chemoreception in perception. Four lines of evidence were presented that support this theory. In the olfactometer bioassays; (1) there was no evidence that ASW avoided the odour blend released by endophyte-infected ryegrass before (AR1 or common-toxic) or after (AR1) plants had been damaged by conspecific insects. In the whole plant choice experiment; (2) there was no evidence that ASW utilised precontact cues (olfaction and vision) to orient away from endophyte-infected (AR1) plants from the outset; (3) ASW showed a strong aversion to endophyte-infected plants with just eight of 45 weevils observed feeding on AR1-infected plants and only one weevil feeding on both hosts during the observational period. In comparison, 32 weevils were observed feeding on endophyte-free plants (4 did not feed); (4) grooming of chemosensory appendages was only observed in weevils enclosed with endophyte-infected plants (AR1 or common-toxic) in the no-choice experiment. This thesis has established a framework, based on investigations of pre- and post- contact behaviour, for investigating mechanisms of insect perception of endophyte and this can be utilised in future studies. Furthermore, this study has identified effects of endophyte on behaviours that have not previously been reported in the endophyte literature and this has provided an exciting area for future research. Keywords: Endophyte, Epichloë festucae variant lolii, perennial ryegrass, Lolium perenne, Argentine stem weevil, Listronotus bonariensis, root aphid, Aploneura lentisci, chemoreception, olfaction, volatile organic compounds, herbivore-induced plant volatiles, gustation, contact chemoreception, post-ingestion, alkaloids, host-selection, host-searching, perception, behaviour. iii Acknowledgements The first people that I would like to thank are my parents, Allen and Glennis, my twin sister Nicole and my brother Shaun. Without your support and encouragement none of this would have been possible. Next, I would like to express my deepest appreciation to Alison Popay, Joanne Jensen and Sarah Finch who have been a key part to making this dream a reality. Thank you for teaching me how to design and carry out experiments, write, vacuum insects and cut grass. It was through your support and encouragement that I had the confidence and ability to pursue a PhD. This PhD was carried out through the BPRC at Lincoln University and AgResearch Ruakura. I was lucky enough to have five supervisors: Dr Alison Popay (AgResearch), Dr Sarah Finch (AgResearch), Professor Michael Rostas (University of Gottingen), Professor Travis Glare (BPRC) and Professor Stephen Goldson (AgResearch and BPRC). Alison, thank you for always making the time to discuss ideas and thoughts with me while giving me the freedom to give it a go and work things out on my own. Sarah, thank you for your support and for helping me with my speling and grammar. I will never start a sentence with the word “But” ever again. Michael, thank you for your assistance with the collection and analysis of volatile compounds. Travis, thank you for providing quick solutions to problems and Stephen, thank you for providing fascinating and truly entertaining discussions on Argentine stem weevil. I would like to thank my ‘second family’ at Plant Protection Ruakura who have made it a joy to come in to work each day. Thanks to your guidance, I no longer add a can of sweet and sour sauce to my lasagna nor do I eat two-minute noodles for my lunch. A special mention to Trevor James who has kept me on my toes with his regular assessments of my fight or flight response. A big thank you to the other members of the endophyte/entomology team - Joanne Jensen, Sarah van Amsterdam, Nikki Webb and Sofia Orre-Gordon for allowing endless discussions on smell, mating behaviour and for keeping me sane. I would like to acknowledge the wider support provided by AgResearch. Firstly, thank you to Vanessa Cave who has been an invaluable source of knowledge on the interesting (and confusing) world of statistics. Thank you for your time, patience and understanding. I truly appreciate your help Vanessa. I would also like to thank Loren Calder, Deirdre Congdon and Megan Symes from the Library and Knowledge Services who kindly sent me hundreds of articles and books. Thank you to Jan Sprosen for running the immunoblot analyses and Derrick Wilson for his assistance with selecting the best (and worst) paddocks for insect collection. Finally, the world’s most wonderful cleaner, Sena Amos, whose infectious laugh and fabulous story telling abilities have kept me entertained for years. Sena, I hope I am still retelling your stories when I am 76. iv This research would not have been possible without the support and financial assistance provided by AgResearch and the BPRC. I am also grateful for a BPRC travel grant which gave me the opportunity to travel to Europe to present my research at an international endophyte conference and visit Wageningen University. I would like to express my thanks to the TR Ellett Agricultural Trust who provided funding towards the operational costs of this PhD. Some of this funding went towards designing and producing olfactometers which will be used in future experiments. This journey started a decade ago when I left high school as an over-eager, super-optimistic, 17-year- old ready to take on the world and pursue a career in science. Ten years later, I have a few fancy pieces of paper and I know a lot about insects and grass making for great work stories. Boom v Table of Contents Abstract ....................................................................................................................................... ii Acknowledgements ..................................................................................................................... iv Table of Contents ........................................................................................................................ vi List of Tables ............................................................................................................................. viii List of Figures .............................................................................................................................