Traditional Diet of the Kiwirrkurra Community Living in the Gibson Desert: Chemical Composition and Functional Characterisation of Identified Foods. Submitted by: Matthew Roland Flavel, BAnVBioSc (Hons) A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy School of Life Sciences College of Science, Health and Engineering La Trobe University Australia January 2018 Acknowledgements: I would like to acknowledge the Kiwirrkurra people and Tjamu Tjamu Aboriginal Corporation for their interest, support and participation throughout this project. I am especially grateful to those who specifically assisted with collection and identification of traditional foods and shared stories and knowledge. I would also like to acknowledge the Wurundjeri people who are the traditional custodians of the land the La Trobe University, Bundoora campus is built on and where I have completed all of my university studies to date. Special thanks to Dr Markandeya Jois who has been my undergraduate lecturer, Honours supervisor, PhD supervisor and mentor in the philosophy and practice of science. His constant stream of ideas, coupled with a deep appreciation for biology and passion for spirited debate has been vital in forging this thesis and my skills as a scientist. Thanks is due to an enormous network of mentors and collaborators who have supported me throughout my candidature. Whilst there is not space to acknowledge all who have helped, certain people need to be mentioned. Dr Alan Yen suggested I study the traditional foods found around Kiwirrkurra and was vital in making this idea a reality. Thanks also to my co-supervisor Dr Adam Mechler and his students for assistance developing the liposome culturing methodology and the use of his laboratory. The chair of my RPP panel Dr Ashley Franks and Elizabeth Mathews for assistance with bacteriological aspect of this work. Professor Weisan Chen and Dr Damien Zanker for assistance assessing the function of particles in axenic media on C. elegans feeding. Dr Daniel Dias and Dr Devin Benheim for assistance with GC-MS analysis. Thanks also to Kate Crossing and Dr Boyd Wright for making the field work component of this thesis a fun and successful experience. Thanks to Dr Jing-Dong Jackie Han, Dr Bo Xian and all members of the PICB, at the Shanghai Institute of Biological Sciences for ongoing collaboration and hospitality whilst I was a visiting student. Thanks also to the other members of the Jois lab who have shared this journey with me and particularly Dr Surafel Tegegne who I have worked closely with along the way. Thanks to Mum, Dad and my family for providing the genes and environment for a healthy and happy life. Thanks also to my wife Shanyn for her patience, support and good humour throughout all my studies, but particularly during my candidature. i TABLE OF CONTENTS Acknowledgements……………………………………………………………………………....……i List of Tables…………………………………………………………………………………….........vi List of Figures……………………………………………………………………………………..….vi Abbreviations and Symbols………………………………………………………………………….ix Thesis Summary………..……………………………………………………………………….........xi Statement of authorship…………………………………………………………………………….xii Audience advice……………………………………………………………….……………………..xii Chapter 1: Literature Review………………………………………………………………………...1 Health gap in Australian Indigenous people………………………………………………..1 Evolutionary Discordance…………………………………………………………………....4 Analysis of the traditional diet of Indigenous Australians………………………………....6 Nutrient composition of Australian foods…….........................................................8 Meat…………………………………………………….……………………………10 Insects…………………………………………………….………………………….13 Plants……………………………………………………….………………………..16 Antioxidants and lifestyle diseases………………………………………………………....23 In vitro studies……………………………………………………………………………….26 Fruits……………………………………………………….………………………..26 Herbs……………………………………………………….………………………..27 Western Diet……………………………………………………….………………………...29 Genetic predisposition……………………………………………….……………………...31 Current diet of Indigenous Australians…………………………….……………………...33 Clinical trials………………………………………………………….……………………..37 Model Organisms…………………………………………………….……………………...39 Caenorhabditis elegans……………………………………….……………………..40 Research direction…………………………………………………….…………………….47 Chapter 2. The bush coconut (scale insect gall) as food at Kiwirrkurra, Western Australia..….48 Abstract……………………………………………………………………………..………..48 Introduction ………………………………………………………………..………………..48 Materials and Methods……………………………………………………..……………….52 ii Study Location…………………………………………………………………….52 Nutrient Analysis………………………………………………………………….55 Results………………………………………………………………………………………56 Classification and Size…………………………………………………………….56 Nutrient Analysis………………………………………………………..………...57 Discussion……………………………………………………………………………...…...59 Chapter 3: Identifying and collection of foods found in the Gibson Desert, Western Australia that are of dietary significance to the Kiwirrkurra people..………………..62 Introduction………………………………………………………………………………...62 Materials and Methods…………………………………………………………………….62 Plant and Insect Material…………………………………………………………62 Results and Discussion…………………………....………………………………….…….63 Food Collection Observations…………....………………………………….…....63 Food Behaviour General Observations...……………………………….….…….74 Chapter 4: Identifying compounds and quantifying antioxidant capacity in traditional foods……………………………………………………………………....77 Introduction……………………………………………………………….……………….77 Materials and Methods……………………….…………………………………….…......778 Plant and insect material…………………………………………………….…...78 Bush food metabolite extraction…………………………………………………79 Polar metabolite derivatization………………………………………………….80 GC-MS Instrument Conditions………………………………………………….80 Data Processing and Statistical Analysis………………………………………..81 Antioxidant Assay………………………………………………………………...81 Results and Discussion…………………………………………………………………….82 In vitro Antioxidant Activity……………………………………………………..82 GC-MS analysis…………………………………………………………………..84 Acylureas………………………………………………………………....86 Amines……………………………………………………………………87 Amino Acids……………………………………………………………...88 Cyclic Polyols…………………………………………………………….90 Dicarboxylic Acids………………………………………………...……..92 iii Fatty Acids………………………………………………………………...93 Fatty Alcohols……………………………………………………………..95 Flavonoids…………………………………………………………………96 Glycosides………………………………………………………………….96 Imidazopyrimidines……………………………………………………….98 Monocarboxylic Acids…………………………………………………….98 Pyridinecarboxylic acids………………………………………………….99 Sugars………………………………………………………………………99 Sugar Acids…………………………………………………………….....100 Sugar Alcohols………………………………………………………..…..101 Steroids……………………………………………………………………101 Triterpenoid………………………………………………………………103 Tricarboxylic acids……………………………………………………….104 Conclusions…………………………………………………………………………………104 Chapter 5. Development of a novel Caenorhabditis elegans model for nutrition studies………107 Abstract……………………………………………………………………………………..107 Introduction………………………………………………………………………………..,107 Materials and Methods…………………………………………………………………….109 C. elegans strains and conditions for growth rate experiments………………..109 Media preparation………………………………………………………………...110 Solubilization of particulate fraction in milk……………………………………111 DNA Extraction and Qualitative Polymerase Chain Reaction…………………112 Qualitative analysis of Bacterial DNA in sample………………………………..112 Quantification of bacterial DNA using real-time quantitative PCR (qPCR).....113 FACS analysis for particle detection……………………………………………..114 Liposome preparation and encapsulation of media……………………………..114 Lifestage Scoring…………………………………………………………………..115 Statistical Analysis………………………………………………………………...115 Results and Discussion…………………………………………………………….116 Chapter 6. Use of a novel C. elegans model to assess bioactivity of Solanum chippendalei……132 Introduction………………………………………………………………………………...129 Materials and Methods…………………………………………………………………….134 iv Plant extract preparation………………………………………………………....134 C. elegans maintenance conditions and growth rate assay conditions…………134 Lifespan assay……………………………………………………………………..135 Media Preparation………………………………………………………………...136 Pumping rate………………………………………………………………………136 Statistical analysis…………………………………………………………………137 Results and Discussion…………………………………………………………………......137 C. elegans growth rate…………………………………………………………….137 C. elegans lifespan…………………………………………………………………140 C. elegans feeding behaviour…………………………………………………….. 144 Chapter 7: General Discussion…………….……...………………………………………………147 References…………………………………….……………………………………………………..153 Appendix A Publications during PhD candidature ……………………….……………………..169 Appendix B: Signed Research agreement with Tjamu Tjamu Aboriginal Corporation…………………………………………………………….…………………..170 Appendix C Ngaanyatjarra council: Permit to enter a reserve……….…………………..179 Appendix D GC-MS chromatograms ……………...................…………….…………………..181 Appendix E Compounds identified by GC-MS in 9 traditional bushfoods.…………..186 Appendix F life stages at 24-hour intervals in tested dietary conditions……………....195 Appendix G Statistical significance of each relative condition in the chapter 5 equivalent growth rate experiments.....................................................................................198 v LIST OF TABLES Table 1.1 Edible native plant foods with Vitamin C content greater than Oranges (Citrus sinensis)………………………………………………………………..22 Table 1.2 Summary of C. elegans lifespan trials on various food extracts……….…..41 Table 2.1 Diameter and weight of bush coconuts collected…….……………………..57 Table 2.2 Calculated values for percentage