Arthropod cuticle: Time-lapse 3D imaging to assess toughening and failure mechanisms A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Science and Engineering. 2019 Daniel Sykes School of Natural Sciences Department of Materials TABLE OF CONTENTS INTRODUCTION: 11 Fracture mechanics of arthropod cuticle 13 Toughening mechanisms in composite materials 15 The use of X-ray computed tomography to investigate microstructure 19 Aims of Papers 1, 2, 3 & 4 22 Alternative format 23 References 25 PAPER 1: Arthropod cuticle: a biological material with diverse mechanical properties 31 PAPER 2: Time-lapse three-dimensional imaging of crack propagation in beetle cuticle 84 PAPER 3: Preservation of mechanical properties in locust tibiae for in situ time-lapse three-dimensional imaging 93 PAPER 4: Effect of hydration on crack propagation in beetle elytra using time-lapse three-dimensional imaging 112 DISCUSSION: 140 Development of a methodology for in situ mechanical testing of hydration-sensitive biological materials 145 Toughness in arthropod cuticle 146 Future work 148 Conclusions 152 References 153 APPENDIX A: Supplementary information 159 Word count: 46,267 2 ABSTRACT Thesis title: Arthropod cuticle: Time-lapse 3D imaging to assess toughening and failure mechanisms Name: Daniel Sykes Institution: University of Manchester Degree Title: Doctor of Philosophy Date: June 2019 This thesis concentrates on one of the least studied mechanical properties of cuticle – toughness. In particular, it investigates how cuticle microstructure impacts crack propagation, and the toughening mechanisms cuticle possesses to protect the vulnerable soft tissues contained within the exoskeleton. I use time-lapse 3D nCT imaging with in situ mechanical tests to investigate toughness and damage progression in arthropod cuticle. In addition, I develop new methodologies of standardised sample preparation and hydration preservation to perform quantitative analysis of fresh locust tibiae and beetle elytra. The results obtained in this thesis have shown that it is possible with these techniques to analyse toughening in fresh and dry cuticles, by qualitative visualisation of the interaction between microstructure and crack propagation and quantitative measurement of toughness values from standardised test samples. It was shown that microstructure is responsible for the numerous extrinsic toughening mechanisms present in cuticle, of which many were previously unreported, and that hydration is responsible for improving the effectiveness and frequency of their occurrence. Furthermore, it was found that the exocuticle contributes little to toughness and that the difference in angle between the crack direction and the fibre orientations of a lamina directly affected the toughening capability of that lamina. To summarise, this thesis displays how a combined approach using state-of-the-art 3D imaging, in situ mechanical testing, sample preparation and hydration preservation techniques can provide us with new insights in how arthropod cuticle shape, microstructure, composition and mechanical properties interact. 3 DECLARATION No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. COPYRIGHT STATEMENT I. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the "Copyright") and she has given the University of Manchester certain rights to use such Copyright, including for administrative purposes. II. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. III. The ownership of certain Copyright, patents, designs trademarks and other intellectual property (the "Intellectual Property") and any reproductions of copyright works in this thesis, for example graphs and tables ("Reproductions"), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. IV. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses. 4 ACKNOWLEDGEMENTS I would like to thank everyone who has helped and supported me during my PhD. Firstly, I would like to thank my supervisors Russell Garwood, Philip Withers and Shelley Rawson for their help and guidance throughout my project, their suggestions for solutions to the problems I encountered and their recommendations on how to improve my experiments, manuscripts and figures. I would also like to thank my colleagues and ex- colleagues Stuart Morse, Julia Behnsen, James Carr, Rob Bradley, Rebecca Hartwell and Chris Egan. During our shared time at the University of Manchester they provided invaluable help, guidance and recommendations. I am very grateful to my parents, Christopher Sykes and Sandra Sykes, and my brother, Gary Sykes, for their continual encouragement and support. The last three and a half years would have been impossible for me without it. I would particularly like to thank my parents for their support from a young age to achieve my ambitions, from waking up in the early hours every day to transport me to the train station so I could go to Greenhead college; to supporting me in my decision to study in Southampton and supporting me throughout my time there; to the continual help, visits and support they have given me during my time at the Natural History Museum and the University of Manchester. I would also like to thank my extended family, my grandma, my uncles and aunties: Tracey, Simon and Lynne, my cousins: Evie, Alice, Karen, Suzanne, Jade, Holly and Nick, and my dearly missed grandparents: Molly, Cyril and Donald. Although I have not been able to spend as much time with them during my PhD as I would have liked, the time I did manage to have was a really appreciated respite from my PhD. 5 Some of my greatest support during my PhD has come from my friendships that started during my undergraduate studies. Benjamin Evans, Rebecca Summerfield, Charlotte Brooks, Kasimir Marks, Anna Mogridge, James Mogridge, Ella Smith and Adam Lomax are great friends who I also wish I had more time with in the last three and a half years, but the time I have had has helped me immeasurably. I developed many lasting friendships during my time at the Natural History Museum that have been a great support to me, but I would like to particularly thank Natasha Vasiliki Almeida. She has been an unwavering support and the similarities between the courses of our PhDs meant her help has been invaluable and vital at all times. I would also like to thank the friends I have made here in Manchester and ones I already had that arrived here before me. Russell Garwood, Charlotte Brassey, James O’Sullivan and all the members of Bill Seller’s lab have made me feel welcome here. I would particularly like to thank Thomas Puschel Rouliez, who has been a great distraction from my PhD by being a true friend and allowing me to have fun and relax once in a while. Without a doubt the most important person during my PhD and in my life is Fernanda Bribiesca Contreras. Since we met in our first year of PhD she has been an undying source of love, support, happiness, kindness and inspiration. She has inspired me to be a better person and scientist by example, and has made my life a happier and more enjoyable one. Even in the toughest times, she has always known how to make me laugh away pain and make everything easier. Muchas gracias Fer por todo, te amo mucho y estoy muy emocionado de pasar mi vida contigo. 6 THE AUTHOR Daniel Sykes Research interests My main research focus is on studying the links between biological forms and structures and their function. I apply computed tomography and advanced 3D image analyses to produce 3D volumetric data of the external form and internal structure of biological materials. Education Ph.D Materials Thesis topic: Arthropod cuticle: Time-lapse 3D imaging to assess toughening and failure mechanisms School of Materials, University of Manchester, UK. Supervisors: Prof. Philip J. Withers, Dr. Russell J. Garwood and Dr. Shelley D. Rawson M.Sci Marine Biology Dissertation topic: Three-dimensional (3D) imaging of polychaete internal anatomy using micro-computed X-ray tomography University of Southampton, UK Supervisor: Dr. Gordon L.J. Paterson Refereed journal publications 1. Paterson GL, Sykes D, Faulwetter S, Merk R, Ahmed F, Hawkins LE, Dinley J, Ball AD, Arvanitidis C. 2014. The pros and cons of using micro-computed tomography in gross and micro-anatomical assessments of polychaetous annelids. Memoirs of Museum Victoria 71:237–46. 2. Reumont BM von, Campbell LI, Richter S, Hering L, Sykes D, Hetmank J, Jenner RA, Bleidorn C. 2014. A Polychaete’s Powerful Punch: Venom Gland Transcriptomics of Glycera Reveals a Complex Cocktail of Toxin Homologs. Genome Biology and Evolution 6:2406–2423. DOI: 10.1093/gbe/evu190. 7 3. Amon DJ, Sykes D, Ahmed F, Copley JT, Kemp KM, Tyler PA, Young CM, Glover AG.