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2014 Randolphclinita BACTERIA INVOLVED IN THE HEALTH OF HONEY BEE (APIS MELLIFERA) COLONIES by Clinita Evette Randolph A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences Summer 2014 © 2014 Clinita Evette Randolph All Rights Reserved UMI Number: 1567820 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI 1567820 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 BACTERIA INVOLVED IN THE HEALTH OF HONEY BEE (APIS MELLIFERA) COLONIES by Clinita Evette Randolph Approved: __________________________________________________________ Diane S. Herson, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Randall L. Duncan, Ph.D. Chair of the Department of Department Biological Sciences Approved: __________________________________________________________ George H. Watson, Ph.D. Dean of the College of Arts and Sciences Approved: __________________________________________________________ James G. Richards, Ph.D. Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS I would like to thank Dr. Herson who has been an amazing advisor and mentor. Dr. Herson’s lab was the perfect lab for me to begin my graduate studies. I am grateful for all the wisdom she has shared with me and I appreciate her encouraging and optimistic attitude, which has helped to keep me driven throughout the project. I have learned so much under her advisement and though many people know I have had a long interest in cancer biology I can honestly say I could not have chosen a better lab to join! I would like to thank my committee members who have provided much guidance for me during the project. Dr. Debbie Delaney has been such a great help in this project from providing honey bees, giving insight on project ideas and analysis, and pushing me to work on her apiary though I am still ironically afraid of bees. Dr. Carlton Cooper has also been a great committee member and mentor. I am grateful for all the wisdom he has shared and for his help from my transition to graduate school to my acceptance to PhD programs. I would also like to thank Caroline Golt who has been a big help with my FAME analysis work. Dr. Jeffry Fuhrmann has also been helpful in allowing me to use Canoco and proving advice on the analysis. I would also like to thank Dr. Eric Wommack for allowing me to use GelCompar. Both of these programs have been a great asset to my research. Katy Evans has also been a big help from letting me sample and collect data with her, providing data for my analysis, and always being willing to answer my bee questions. iii Thank you to all the Herson lab members who have also been a tremendous help. Mollee was a great example for me and always encouraging while I transitioned into graduate school and still continues to be just as supportive and helpful. I would like to thank Paul for training me in much of the molecular work and for his help in the apiary. I am not sure how I would have gotten this far without him. I would also like to thank Dr. Russell Vreeland who not only was my first biology lab professor at West Chester University, but also began this project with Paul that I have been able to take on as a master’s student. I would like to thank Julia and Kalli who helped with the antibiotic susceptibility testing and PCR-DGGE respectively. They both have been a big help in the project. I also would like to James and Beth who have also been supportive throughout my time at the University of Delaware. I would like to thank my family and friends who have been a great support. My parents are my biggest supporters and have been encouraging from grade school to graduate school that I can reach my goals and excel. My sisters Christina and Mildred are also supportive of me and have been a big source of help and encouragement during graduate school. I would also like to thank my aunts, uncles, cousins, friends, and church family who always encourage me to keep going. Above all I would like to thank God who has blessed me with the opportunity to begin and complete my graduate studies at the University of Delaware. I believe with God nothing is impossible (Matthew 19:26) and I know I could not have done this without Him. iv TABLE OF CONTENTS LIST OF TABLES ...................................................................................................... viii LIST OF FIGURES ....................................................................................................... ix ABSTRACT .................................................................................................................. xi Chapter 1 INTRODUCTION .............................................................................................. 1 1.1 Apis mellifera ................................................................................................ 1 1.2 Honey bee Decline ....................................................................................... 2 1.3 Bacteria associated with Apis mellifera ........................................................ 3 1.4 Bee Bread ..................................................................................................... 4 1.5 Antibiotics .................................................................................................... 5 1.5.1 Oxytetracycline Hydrochloride ........................................................... 6 1.5.2 Streptomycin ........................................................................................ 7 1.5.3 Tylosin Tartrate ................................................................................... 7 1.5.4 Fumagillin-B ........................................................................................ 8 1.5.5 Ampicillin ............................................................................................ 8 1.6 Hypothesis .................................................................................................... 9 2 MATERIALS AND METHODS ..................................................................... 11 2.1 Strains ......................................................................................................... 11 2.1.1 Bee Bread Sources and Sampling ...................................................... 11 2.1.2 Bacterial Isolation from Bee Bread ................................................... 11 2.1.3 Bacterial Isolations ............................................................................ 12 2.2 Media .......................................................................................................... 12 2.2.1 Agar Supplemented Media ................................................................ 12 2.2.2 Gellan Gum Supplemented Media .................................................... 12 2.2.3 Low Nutrient Media .......................................................................... 13 2.3 DNA Isolation ............................................................................................ 13 v 2.3.1 Bee Bread DNA Isolation .................................................................. 13 2.3.2 Bacterial DNA Isolation .................................................................... 13 2.3.3 Bacterial DNA Ethanol Precipitation ................................................ 14 2.4 Denaturing Gradient Gel Electrophoresis .................................................. 14 2.4.1 Polymerase Chain Reaction (PCR) of 16S RNA Gene ..................... 14 2.4.2 Denaturing Gradient Gel Electrophoresis ......................................... 16 2.5 Fatty Acid Methyl Ester (FAME) Analysis for Identification of Aerobes and Actinomycetes ..................................................................................... 16 2.6 WalkAway® 40 System for Identification of Enterobacteriaceae ............ 18 2.7 Antibiotic Susceptibility Testing ................................................................ 19 2.8 Statistical Analysis ..................................................................................... 20 2.8.1 Denaturing Gradient Gel Electrophoresis ......................................... 20 2.8.2 Fatty Acid Methyl Ester Analysis ..................................................... 22 3 RESULTS ......................................................................................................... 23 3.1 Bacterial Isolation from Bee Bread ............................................................ 23 3.2 Bee Bread DNA Isolation ........................................................................... 23 3.3 DGGE Ladder ............................................................................................. 24 3.4 Denaturing Gradient Gel Electrophoresis .................................................. 25 3.5 FAME for the Identification of Bacteria .................................................... 29 3.6 WalkAway® 40 for the Identification of Enterobacteriaceae Organisms.
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