DOCTOR OF PHILOSOPHY Exploiting Entomopathogenic Nematode Neurobiology to Improve Bioinsecticide Formulations Morris, Rob Award date: 2020 Awarding institution: Queen's University Belfast Link to publication Terms of use All those accessing thesis content in Queen’s University Belfast Research Portal are subject to the following terms and conditions of use • Copyright is subject to the Copyright, Designs and Patent Act 1988, or as modified by any successor legislation • Copyright and moral rights for thesis content are retained by the author and/or other copyright owners • A copy of a thesis may be downloaded for personal non-commercial research/study without the need for permission or charge • Distribution or reproduction of thesis content in any format is not permitted without the permission of the copyright holder • When citing this work, full bibliographic details should be supplied, including the author, title, awarding institution and date of thesis Take down policy A thesis can be removed from the Research Portal if there has been a breach of copyright, or a similarly robust reason. 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Oct. 2021 QUEEN’S UNIVERSITY BELFAST INSTITUTE FOR GLOBAL FOOD SECURITY SCHOOL OF BIOLOGICAL SCIENCES Exploiting Entomopathogenic Nematode Neurobiology to Improve Bioinsecticide Formulations ROBERT MORRIS Degree in Biology, Liverpool John Moores University, U.K. FOR THE DEGREE OF DOCTOR OF PHILOSOPHY September 2019 Exploiting Entomopathogenic Nematode Neurobiology to Improve Bioinsecticide Formulations I Exploiting Entomopathogenic Nematode Neurobiology to Improve Bioinsecticide Formulations Robert Morris A dissertation submitted to Queen’s University Belfast in accordance with the requirements for award of the degree of PhD in the Institute for Global Food Security, School of Biological Sciences, September 2019. Word Count: 34,225 II Abstract Entomopathogenic nematodes (EPNs) are soil dwelling organisms that target and kill insects. They have sophisticated chemosensory apparatus, with which they perceive host associated cues to locate them. Once a suitable host is located, they employ a diverse range of host finding behaviours to facilitate contact with a host, a prerequisite for infection. These EPNs are described as beneficial nematodes as they are employed as biocontrol agents for crop protection against insect pests. Insects cause devastating damage to annual crop yields. Many chemical pesticides have been banned across the world due to their devastating effects on the environment and the health of animals, therefore, a safer alternative is essential to protect crops from pests. EPNs have proven that they can provide effective protection against insects in both field and glasshouse trials, however, there have been reports of inconsistencies in biocontrol efficiency. This could be attributed to our limited knowledge of their biology post-application, and the molecular mechanisms that drives their host finding behaviours. Some insight into the genes that regulate these diverse host finding behaviours in EPNs could be used to enhance their biocontrol efficiency. Small interfering RNAs can silence genes post- transcriptionally. One method to achieve gene knockdown with small interfering RNAs is RNA interference (RNAi). RNAi is a powerful tool to determine the function of a gene, as it can be tailored to target a specific gene of interest, silence the gene in vivo, and observe the effect this has on the organism. Functional studies using RNAi have identified the role of many genes in the model organism Caenorhabditis elegans, including many genes involved in sensory perception. In this study, the neuropeptide encoding gene flp-21, will be targeted using RNAi in Steinernema carpocapsae to observe its effect on their host finding behaviours. This study is the first instance of RNAi in a Steinernema species of EPN, where Sc-flp-21 was suppressed by 84 % (P<0.0001) by soaking S. carpocapsae IJs in a high amount of dsRNA for 48 hours with 50 mM serotonin. The suppression of Sc-flp-21 had significant effects on their host finding behaviours through impeding olfactory perception in these nematodes. Another method used to identify genes that regulate intraspecific III phenotypic differences between populations is RNA-sequencing. Sequencing the transcriptome allows observation of the gene expression pattern of a single cell or entire organism at one time. Differentially expressed genes between phenotypically different populations can indicate the genes responsible for the intraspecific differences observed between populations. Transcriptome analysis identified differentially expressed genes between S. carpocapsae strains that showed phenotypic differences with regards to their host finding behaviours. Nictation behaviour in S. carpocapsae may be heavily regulated by Sc-daf-2 and Sc-daf-7, which has been described in C. elegans previously. Host finding behaviour was enhanced in Breton strain, and transcriptome analysis revealed many GPCRs and neuropeptides to be upregulated in Breton, relative to the other two strains. Functional studies would need to take place before these genes’ roles can be confirmed. Natural EPN isolates were collected from Northern Irish soil, and challenged through host finding assays, along with various other trait assessments, to investigate their natural diversity. Ten isolates were collected, spanning at least four EPN species: S. feltiae, S. affine, Heterorhabditis downesi and other, unidentified Heterorhabditis species. All isolates tested, including some laboratory strains, showed phenotypic variation in their host finding behaviours. Intraspecific phenotypic variation was also observed between S. feltiae strains. The large phenotypic variation observed in natural populations of EPN may allow biocontrol formulations to be tailored to the user’s needs, and facilitate artificial selection for enhanced EPN strains. This thesis focuses on the molecular basis of host finding behaviours in EPNs, and exploring their natural diversity to enhance their biocontrol efficiency for crop protection. IV Dedication and Acknowledgments I would like to dedicate this Thesis to my Grandmother, who was always my biggest supporter and inspiration in life. Firstly I would like to thank Queen’s University Belfast and their Business Alliance funding for allowing me to undertake and carry out my PhD degree. I am very grateful for my supervisors, Dr Johnathan Dalzell and Prof Aaron Maule, for the guidance, time and effort they’ve provided since starting my PhD, and for their academic expertise and knowledge, which helped me become a better scientist. Working in the same laboratory and starting my PhD alongside Matthew Sturrock was a blessing, his humour, professional and personal support was second to none, and I am very thankful to call him a close friend. I would like to especially thank Dr Debbie Cox for her encouragement, teachings, and for providing both professional and emotional support when I needed it. A big thank you to, Dr Neil Warnock, Dr Leonie Wilson and Dr Ciaran McCoy for their help, advice and all the good times we shared together since the moment we met. Dr Nikki Marks provided me with support and strength when I needed it, and brightened up my day every day we had a catch up, which I am so grateful for. Thank you to my family and friends for their patience, understanding and support during my PhD experience. To my new found family in Belfast, I thank Erica, CJ and Ben for making my time there so special; I couldn’t have done it without your support. I would also like to express my gratitude to the many others that also supported me during my time in Belfast, thank you for making my experience so unforgettable. And finally, thank you to Catherine Maciver and John Parker for always believing in me and supporting me throughout our friendship. V Author’s Declaration I declare that the work in this thesis was carried out in accordance with the requirement of Queen’s University Belfast’s Regulations and Code of Practise for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate’s own work. Work done in collaboration with, or with the assistance of others, is indicated as such. Any views expressed in the dissertation are these of the author. Signed ………………………………………………………………. Date……………………………. VI Table of Contents Title ................................................................................................................. I Cover page ..................................................................................................... II Abstract ......................................................................................................... III Dedication and Acknowledgments ...............................................................