Foraging behaviour of female Weddell seals (Leptonychotes weddellii) during lactation: new insights from dietary biomarkers A thesis submitted in partial fulfilment of the requirements for the degree in Doctor of Philosophy in Antarctic Studies by Crystal C. Lenky November 2012 Acknowledgements Only one name appears on the cover of a thesis, but there are many people involved in its creation. First and foremost, I thank my supervisors for their endless encouragement, knowledge and insightful conversations throughout this journey. I thank Dr Regina Eisert for instilling in me an appreciation of the analytical method and her expertise on data analysis; Dr Victoria Metcalf for her friendship, enthusiasm and advice on all aspects of life; Dr Michael Lever for his wealth of knowledge on betaines and for providing me with lab and office space after the February 22 earthquake; Dr Olav Oftedal for his expertise in nutrition and guidance on scientific writing; Dr Marie Squire for her guidance on the NMR assays, and Professor Bryan Storey for providing me with a great working environment. Funding for this thesis was provided by a National Science Foundation, Office of Polar Programs grant to Drs Regina Eisert and Olav Oftedal. Many people have also given their time and expertise in assisting me with fieldwork, laboratory and data analysis. I thank Dr Wendy Hood, Lisa Ware, Warren Lynch and Rich Joss for their assistance in the field and for making my time in Antarctica an enjoyable experience. Michael Jakubasz (Smithsonian National Zoological Park) provided invaluable assistance during my stay at the Nutrition Lab. I thank him for his guidance on the proximate composition assays and for sorting permits and shipping details. I owe a huge thank you to Chris McEntyre (Canterbury Health Laboratories) for his friendship, and his assistance with the LC-MS/MS method development. I thank Dr Matt Pinkerton (NIWA, New Zealand), Dr Gretchen Hoffman (UC Santa Barbara, USA), Dr Art DeVries (University of Illinois, Urbana-Champaign, USA), Victoria Metcalf, Regina Eisert and Olav Oftedal for providing all of the samples that were used in this thesis. The Gateway Antarctica community provided support and humour over the last four years; especially my office mates Briar Wait, Erin Nuefeld and Oliver Marsh. I thank my friends, both old and new, for supporting me on this journey. Lastly, I thank my family for believing in me and allowing me to follow my dreams, even if they did take me thousands of miles away. This thesis is dedicated to Charlotte and Bernard Lenky. ii Table of Contents Acknowledgements .................................................................................................................. ii Table of Contents ................................................................................................................... iii List of Tables ........................................................................................................................... ix List of Figures .......................................................................................................................... xi List of Abbreviations ........................................................................................................... xvii Abstract .................................................................................................................................. xix Chapter One ............................................................................................................................. 1 Introduction and Literature Review ...................................................................................... 1 1.1 Brief overview ................................................................................................................. 2 1.2 Methods for studying the diet of marine mammals ......................................................... 3 1.2.1 Stomach and faecal analysis ..................................................................................... 3 1.2.2 Molecular analysis .................................................................................................... 5 1.2.3 Stable isotope analysis .............................................................................................. 5 1.2.4 Fatty acid signature analysis ..................................................................................... 6 1.3 Pinniped lactation strategies ............................................................................................ 7 1.4 Foraging in lactating Weddell seals ................................................................................. 8 1.5 The biomarker method ................................................................................................... 12 1.5.1 Marine osmolytes .................................................................................................... 12 1.5.2 Osmolytes as dietary biomarkers ............................................................................ 12 1.5.3 Trimethylamine N-oxide (TMAO) ......................................................................... 14 1.5.4 Glycine betaine (GB) .............................................................................................. 17 1.5.5 Arsenobetaine (AsB) ............................................................................................... 18 iii 1.5.6 Dimethylsulfoniopropionate (DMSP) ..................................................................... 21 1.5.7 Homarine ................................................................................................................ 24 1.6 Thesis aims and research hypotheses ............................................................................. 26 Chapter Two ........................................................................................................................... 29 Development and validation of a method for measuring dietary biomarkers in marine organisms using 1H NMR spectroscopy ............................................................................... 29 2.1 Introduction .................................................................................................................... 30 2.1.1 Nuclear magnetic resonance spectroscopy ............................................................. 30 2.1.2 Application of 1H NMR to studying dietary biomarkers ....................................... 32 2.2 Methods .......................................................................................................................... 33 2.2.1 Materials ................................................................................................................. 33 2.2.2 Sample preparation ................................................................................................. 34 2.2.3 1H NMR analysis and spectral processing .............................................................. 36 2.2.4 Precision and recovery ............................................................................................ 38 2.2.5 Limit of detection (LOD) and limit of quantitation (LOQ) .................................... 39 2.3 Results ............................................................................................................................ 41 2.3.1 Biomarker identification and quantification ........................................................... 41 2.3.2 Precision and recovery ............................................................................................ 46 2.3.3 LOD and LOQ ........................................................................................................ 47 2.4 Discussion ...................................................................................................................... 49 Chapter 3 ................................................................................................................................ 53 Development and validation of a method for detecting dietary biomarkers in serum and tissues using liquid chromatography – tandem mass spectrometry .................................. 53 3.1 Introduction .................................................................................................................... 54 3.1.1 Liquid chromatography-tandem mass spectrometry (LC-MS/MS) ........................ 54 iv 3.1.2 Application of LC-MS/MS to measuring dietary biomarkers ................................ 57 3.2 Materials ........................................................................................................................ 58 3.2.1 Reagents .................................................................................................................. 58 3.2.2 Internal standard synthesis ...................................................................................... 58 3.3 Prey – method validation ............................................................................................... 60 3.3.1 Preliminary experiments ......................................................................................... 60 3.3.2 Sample preparation ................................................................................................. 62 3.3.3 LC-MS/MS instrumentation ................................................................................... 63 3.3.4 Calibration curve ..................................................................................................... 66 3.3.5 Instrument validation .............................................................................................