CHITINASE EXPRESSION IN THE STOMACH OF THE AYE-AYE (DAUBENTONIA MADAGASCARIENSIS). A thesis submitted To Kent State University in partial Fulfilment of the requirements for the Degree of Master of Arts By Melia G. Romine August 2020 © Copyright All rights reserved Except for previously published materials Thesis written by Melia G. Romine B.S., Kent State University, 2018 M.A., Kent State University, 2020 Approved by _______________________, Advisor Dr. Anthony J. Tosi _______________________, Chair, Department of Anthropology Dr. Mary Ann Raghanti _______________________, Interim Dean, College of Arts and Sciences Dr. Mandy Munro-Stasiuk TABLE OF CONTENTS -----------------------------------------------------------------------------------iii LIST OF FIGURES ------------------------------------------------------------------------------------------v LIST OF TABLES -------------------------------------------------------------------------------------------vi DEDICATION ----------------------------------------------------------------------------------------------vii ACKNOWLEDGEMENTS ------------------------------------------------------------------------------viii CHAPTERS I. INTRODUCTION ------------------------------------------------------------------------------1 FEEDING STRATEGIES ---------------------------------------------------------------------2 OPTIMAL FOOD SOURCES ----------------------------------------------------------------3 BODY SIZE -------------------------------------------------------------------------------------6 DAUBENTONIA MADAGASCARIENSIS AND THEIR ADAPTIVE SUITE----------8 DENTITION -------------------------------------------------------------------------------------9 SPECIALIZED MIDDLE DIGIT-------------------------------------------------------------9 AUDITORY SYSTEM -----------------------------------------------------------------------10 BEHAVIOR AND ECOLOGY OF DAUBENTONIA MADAGASCARIENSIS -------11 NATURAL HISTORY OF DAUBENTONIA MADAGASCARIENSIS ----------------15 PHYLOGENY OF DAUBENTONIA MADAGASCARIENSIS --------------------------15 COMPARATIVE PRIMATE: EULEMUR MACACO -----------------------------------15 CHITIN AND CHITINASE ------------------------------------------------------------------17 HYPOTHESIS ---------------------------------------------------------------------------------19 II. METHODS -------------------------------------------------------------------------------------21 SAMPLES --------------------------------------------------------------------------------------21 RNA SEQUENCING -------------------------------------------------------------------------22 iii SEQUENCE ALIGNMENTS AND TRIMMING ----------------------------------------23 BIOINFORMATIC TOOLS FOR ANALYSIS -------------------------------------------24 III. RESULTS --------------------------------------------------------------------------------------27 QUALITY REPORT OF SEQUENCE READS-------------------------------------------27 ANNOTATION REPORTS ------------------------------------------------------------------27 ABUNDANCE REPORTS -------------------------------------------------------------------32 STATISTICAL ANALYSIS -----------------------------------------------------------------33 EVOLUTIONARY COMPARISONS OF CHIA IN PRIMATES ----------------------34 IV. DISCUSSION AND CONCLUSION ------------------------------------------------------37 DISCUSSION ----------------------------------------------------------------------------------37 FUNCTIONAL CHITINASE IN AYE-AYE AND BLACK LEMUR ----------------37 AYE-AYE AND CHITINASE ORIGINS -------------------------------------------------39 RE-EXAMINING THE JARMAN-BELL PRINCIPLE / KAY’S THRESHOLD ---39 CHITINASE AND INSECT EATING HUMANS ---------------------------------------40 CONCLUSION AND FUTURE CONSIDERATIONS ----------------------------------40 REFERENCES ----------------------------------------------------------------------------------------------42 iv LIST OF FIGURES Figure 1. Image of aye-aye ---------------------------------------------------------------------------------2 Figure 2. Table adapted from “A Jarman-Bell Model of Primate Feeding Niches” ----------------4 Figure 3. Image of Japanese Walnut ----------------------------------------------------------------------5 Figure 4. Map distribution of aye-aye sightings --------------------------------------------------------11 Figure 5. Phylogeny of Strepsirrhines--------------------------------------------------------------------16 Figure 6. 3-D rope model ----------------------------------------------------------------------------------17 Figure 7. Example of de novo assembly -----------------------------------------------------------------23 Figure 8. Top 10 CHIA1 matches to aye-aye -----------------------------------------------------------29 Figure 9. Top 10 CHIA2 matches to aye-aye -----------------------------------------------------------29 Figure 10. Top 10 CHIA3 matches to aye-aye ---------------------------------------------------------29 Figure 11. Top 10 CHIA4 matches to aye-aye ---------------------------------------------------------30 Figure 12. Top 10 CHIA5 matches to aye-aye ---------------------------------------------------------30 Figure 13. Top 10 CHIA1 matches to black lemur ----------------------------------------------------30 Figure 14. Top 10 CHIA2 matches to black lemur ----------------------------------------------------30 Figure 15. Top 10 CHIA3 matches to black lemur ----------------------------------------------------31 Figure 16. Top 10 CHIA4 matches to black lemur ----------------------------------------------------31 Figure 17. Top 10 CHIA5 matches to black lemur-----------------------------------------------------31 Figure 18. Top 10 GAPDH matches to aye-aye --------------------------------------------------------32 Figure 19. Top 10 GAPDH matches to black lemur ---------------------------------------------------32 Figure 20. Chitinase catalytic site ------------------------------------------------------------------------34 Figure 21. Chitin-binding domain ------------------------------------------------------------------------35 Figure 22. Phylogenetic Tree -----------------------------------------------------------------------------36 v LIST OF TABLES Table 1. Overall concept of Jarman-Bell Principle -----------------------------------------------------6 Table 2. Table adapted from "Testing Convergent Evolution in Auditory Processing-----------11 Table 3. Notable observational studies -----------------------------------------------------------------14 Table 4. Stomach tissue samples ------------------------------------------------------------------------21 Table 5. Quick Biology sequencing identifiers --------------------------------------------------------23 Table 6. Kallisto scripts ----------------------------------------------------------------------------------25 Table 7. Gene and Protein IDs----------------------------------------------------------------------------26 Table 8. Sequencing Report Summary -----------------------------------------------------------------27 Table 9. Transcript per million values for aye-aye ---------------------------------------------------32 Table 10. Transcript per million values for black lemur ---------------------------------------------33 Table 11. CHIA to GAPDH values ---------------------------------------------------------------------33 vi Dedication I dedicate this project to my beloved mother, Martha Renee Romine. You have always seen my potential even when I could not. You are my rock and I love you always. Also, to my official cheerleaders, twisted sistas, confidants, and everything else, Rikayla Wright and Arrik Brown vii Acknowledgements First and foremost, I would like to thank Dr. Anthony Tosi for giving me the opportunity to pursue my master’s degree. Being in this program has opened up the door for new opportunities. Next, I would like to thank my committee members, Dr. Mary Ann Raghanti and Dr. Richard Meindl. I appreciate your expertise through the program. Then I would like to take the time to recognize my lab mates and faculty members that helped me through this whole process. Morgan Chaney, you have taken so much time to teach me the foundational techniques I needed for this project. I consider you a mentor and hope to continue to work with you on future projects. You are destined to be great! Cody Ruiz, thank you for always lending an ear and talking me off the ledge so to speak. I will always treasure our friendship. Danielle Jones, you have always been the voice of reason throughout this program and I could not have gotten this far without you. Dr. Metin Eren, Dr. Owen Lovejoy, and Dr.Linda Spurlock, you all have always believed in me from the start. Your encouraging words made me realize that I do belong in the field of science. Dr. Sangeet Lamichhaney, I appreciate you for taking time out of your schedule to guide me through all the bioinformatics required for my project. You made it easy to learn and I will carry that with me throughout my entire career. Finally, lastly but certainly not least, thank you to all my cohort! Special thanks to Dusty, Sarah, Alyssa and Anna for forming the official pork and stouts crew. You all dragged me to the finish line whether I wanted to cross it or not. You all are friends I will have for the rest of my life. viii Chapter 1: Introduction The biological success of an organism is measured by the amount of offspring they produce. Adequate nutrition and caloric intake are critical for