Assessing the Presence of Chitinases in the Digestive Tract and their Relationship to Diet and Morphology in Freshwater Fish Word count: 25 157 Andy Vervaet Student number: 01000500 Supervisor: Prof. dr. Ir. Geert Janssens Supervisor: Dr. Arturo Muñoz Saravia A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Veterinary Medicine Academic year: 2018 - 2019 Ghent University, its employees and/or students, give no warranty that the information provided in this thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result in any infringement of third-party rights. Ghent University, its employees and/or students do not accept any liability or responsibility for any use which may be made of the content or information given in the thesis, nor for any reliance which may be placed on any advice or information provided in this thesis. ACKNOWLEDGEMENTS Writing this work would not have been possible without all the guidance, help and motivation I received during the process. I offer my sincerest gratitude to all the people who played a role in finishing the thesis. First, I would like to thank both my promotors; prof. dr. ir. Geert Janssens and dr. Arturo Muñoz Saravia. For offering me the opportunity to work on this exciting subject and allowing me to get an invaluable experience of learning to work in challenging conditions abroad. Their guidance and expertise on different issues were integral to finishing this work. Secondly, I would like to thank all the people who helped me during certain parts of the study; the people at IDP-Pacu who made their lab facilities for me in Bolivia, Donna Vanhauteghem for helping me to process my samples, and Stefania Magnusdottir and Joanna Wolthuis of UMC Utrecht for performing the data analysis of the DI-MS samples. I especially thank my guest family, for providing me with shelter and food during the sampling period on Isla del Luna. Thank you, Esperenza, for making me feel part of your family, your hospitality and always positive attitude is missed. Thirdly, I thank all the friends I made during my studies for their moral support as well as the fun distractions they provided when taking a break from my research. I want to single out two of my best friends, Daphné Van der Eycken and Matthieu Salamone. They were of great aid in deliberating my findings and generally a big source of motivation and support, throughout. Additionally, Matthieu Salamone, probably the best data scientist in the world, is thanked for his crucial help in statistics and the interpretation thereof. Last, but certainly not least, I would like to thank my parents for their unwavering faith in me and the unconditional support they have given me throughout my studies. 3 TABLE OF CONTENTS List of abbreviations ................................................................................................................................................. 5 Summary ................................................................................................................................................................. 6 Samenvatting ........................................................................................................................................................... 7 1. Literature review .............................................................................................................................................. 8 1.1. Introduction ............................................................................................................................................ 8 1.2. Chitin ...................................................................................................................................................... 9 2D-structure ................................................................................................................................... 9 3D-structure and crystallography ................................................................................................... 9 Chemical properties .................................................................................................................... 10 Function and distribution ............................................................................................................. 11 1.3. Chitinase .............................................................................................................................................. 11 Nomenclature and classification .................................................................................................. 11 Distribution .................................................................................................................................. 14 1.4. Anatomy of the fish gastrointestinal system ......................................................................................... 16 Headgut ....................................................................................................................................... 17 Foregut ........................................................................................................................................ 17 Midgut.......................................................................................................................................... 17 Hindgut ........................................................................................................................................ 18 Liver ............................................................................................................................................ 18 Pancreas ..................................................................................................................................... 18 1.5. Digestion of carbohydrates .................................................................................................................. 19 Physical processes ...................................................................................................................... 19 Chemical processes .................................................................................................................... 19 Enzymatic processes .................................................................................................................. 19 Microbial processes ..................................................................................................................... 20 Absorption ................................................................................................................................... 20 1.6. Fish chitinases ..................................................................................................................................... 21 Origin of chitinase activity in the fish gut...................................................................................... 21 Role of fish chitinases ................................................................................................................. 23 Nutritive role of the chitinolytic system ........................................................................................ 24 Chemical disruption ..................................................................................................................... 29 Immunity ...................................................................................................................................... 29 Link to morphometry .................................................................................................................... 30 2. The aim of the study ...................................................................................................................................... 30 2.1. Objective 1: Gut morphology and morphometry ................................................................................... 30 2.2. Objective 2: Stable Isotope Analysis. ................................................................................................... 31 2.3. Objective 3: Metabolomics ................................................................................................................... 31 2.4. Objective 4: Gene expression. ............................................................................................................. 31 3. Material and methods .................................................................................................................................... 31 3.1. Study site and fish collection ................................................................................................................ 31 3.2. Morphometric data of the investigated fish ........................................................................................... 31 3.3. Statistical analysis of morphometric data ............................................................................................. 32 3.4. Stable isotope analysis [SIA] ................................................................................................................ 33 3.5. Metabolomics ....................................................................................................................................... 33 3.6. Gene expression .................................................................................................................................. 34 4. Results ..........................................................................................................................................................