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3 Fatty Acid Profile of the Clover Root Weevil http://researchcommons.waikato.ac.nz/ Research Commons at the University of Waikato Copyright Statement: The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). The thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: Any use you make of these documents or images must be for research or private study purposes only, and you may not make them available to any other person. Authors control the copyright of their thesis. You will recognise the author’s right to be identified as the author of the thesis, and due acknowledgement will be made to the author where appropriate. You will obtain the author’s permission before publishing any material from the thesis. Seasonal Comparison of Lipid Composition and Metabolism in Parasitised and Non-Parasitised Clover Root Weevil (Sitona lepidus) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at The University of Waikato by JOLENE MARY BROWN 2014 Abstract Abstract Since its arrival in New Zealand the clover root weevil (Sitona lepidus (syn. flavescens) (Coleoptera: Curculionidae) (CRW) has caused serious damage to New Zealand’s agricultural sector. The introduction of the biocontrol agent, Microctonus aethiopoides has caused a significant reduction in the population of CRWs. During their research on the CRW, AgResearch scientists discovered that the abdominal fat body and lipids present in the haemolymph in adult CRW varied with season, sex, insect age and parasitism. Parasitism has been reported to change the lipid composition of other species of insects. The purpose of the present study was to compare the chemical composition of the lipids present in parasitised and non-parasitised CRW adults, and determine how these lipids change with physiological state and parasitoid development. The investigation into how M. aethiopoides initiates these changes was extended to examine the roles of juvenile hormones and teratocytes in lipid regulation. A one-step method of extraction and derivatisation was created to determine the fatty acid profile of CRWs. This method gave higher recovery percentages and higher reproducibility than the traditional two-step methods that were trialled, and enabled individual CRWs to be analysed. The fatty acid profile of the CRW was similar to that of other insects reported containing mainly the 16 carbon saturated and monounsaturated fatty acids and the 18 carbon saturated, monounsaturated and polyunsaturated fatty acids. The one-step method was also used to track differences in the fatty acid profiles of individual CRWs with differing sex, age, parasitism and physiological state. The fatty acid profiles of male and female CRWs were similar, with only significant differences between the concentrations of the 16:1 and 18:0 fatty acids. Due to variation between individual samples in the entire sample set, no obvious correlations were found between physiological state and fatty acid composition, or distinctions between state of parasitism and fatty acid profile. Basic statistical methods were utilised initially, however, the complexity of the data set required ii Abstract multivariate analysis. PCA, LDA and QDA were utilised but again no correlations or distinctions were found when the whole sample set was analysed. Results from the Gisborne subset, collected from the same location and at the same time, had reduced variation between individuals, and this allowed some distinctions to be made between parasitised and non-parasitised samples. Teratocytes are cells that have dissociated from the serosal membrane that occur in the haemolymph of CRWs that have been parasitised by M. aethiopoides. The fatty acid composition of these cells was investigated using the one-step method and a MALDI-TOF spectrometry method, which detects triacylcglycerols. The fatty acid profile of teratocytes was not significantly different to that of the CRW. Juvenile hormones (JHs) control postembryonic development and adult reproduction. They are present in all insects and JH III is the most common of the six possible JHs. The LC-MS method of Miyazaki et al was modified and this allowed for the determination of JH III within samples of 50 CRW adults. The comparison between parasitised and non-parasitised samples found that parasitised samples had significantly higher levels of JH III than did their non- parasitised counterparts. iii “I am neither especially clever nor especially gifted. Acknowledgements Acknowledgements I am so grateful to everyone that has helped make this thesis possible. Firstly I would like to express immense gratitude to my primary supervisor Associate Professor Merilyn Manley-Harris. Her professional support and continued friendship throughout this project has been incredible. Few students are fortunate enough to have a supervisor who has enough faith in them to allow them to work independently, yet is always available with helpful advice. It is little wonder why Merilyn has so many students. Thanks to Dr. Pip Gerard, my AgResearch supervisor, for the project itself and the continued help and support throughout my work. I have thoroughly enjoyed working with you and the team at AgResearch, and am so grateful for all of the time and expert help that I have received. Thanks to my third supervisor Professor Alistair Wilkins. Your advice and expertise was always on hand and I am especially grateful for your help developing my GC-MS method. I must also say a big thank you to Dr. Ray Littler for the countless hours he helped me. I hope that I taught you as much about weevils as you taught me about statistics! To Lily and Jess, the two undergraduates that I had the privilege of working with, thank you very much for all of your help. Thanks also to all of the department’s experts who helped me with the instruments that I needed to use. To Wendy, Jenny, Jonathan, Meenashki and Maria - I really appreciated your help. Thank you to everyone in the chemistry department at The University of Waikato, I have really enjoyed my time here. Thanks for providing friendly faces, and constant sources of advice and conversation. Thanks especially to those in E3.24; I think that we will go down as the coolest lab in history. iv Acknowledgements I would like to thank my parents, my family and Hadleigh for their love and support. Thanks also to my friends and flatmates for helping me keep my sanity and providing never-ending encouragement. Lastly I would like to acknowledge the scholarships that I have been fortunate to receive - Top Achiever Doctoral Scholarship, Shirtcliffe Fellowship, Waikato Graduate Womens’ Trust Merit Award for Doctoral Study and the Royal Society of New Zealand Travel Grant. v Table of Contents Table of Contents Abstract ...................................................................................................................... ii Acknowledgements ................................................................................................... iv Table of Contents ...................................................................................................... vi List of Tables.......................................................................................................... xvii List of Figures ........................................................................................................ xxv List of Abbreviations.......................................................................................... xxxvii 1 Introduction .................................................................................................... 1 1.1 The Significance of Clover Root Weevil in New Zealand .......................... 1 1.1.1 Agriculture in New Zealand and the Importance of Clover ............... 1 1.1.2 The Clover Root Weevil (Sitona lepidus) .......................................... 1 1.1.3 Establishment of the Clover Root Weevil within New Zealand ........ 2 1.1.4 Control Options .................................................................................. 3 1.1.5 Possible Parasitoids Already in New Zealand .................................... 4 1.1.6 The Search for a Biological Control Agent and the Discovery of Microctonus aethiopoides .............................................................. 5 1.1.7 Microctonus aethiopoides .................................................................. 6 1.1.8 Introduction of Microctonus aethiopoides ......................................... 7 1.1.9 Success of Microctonus aethiopoides ................................................ 8 1.2 Background to Project ................................................................................. 8 1.2.1 Introduction ........................................................................................ 8 1.2.2 Context of the Project ......................................................................... 9 vi Table of Contents 1.2.3 General Purpose of Project ............................................................... 11 1.2.4 Specific Objectives ........................................................................... 12 1.3 Scope and Limitations of Project .............................................................. 13 2 Method Development ................................................................................... 15 2.1 Introduction ............................................................................................... 15 2.1.1 Sample Extraction ...........................................................................
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