Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2017 Termite Gut Microbes as Tools and Targets for Termite Control Chinmay Vijay Tikhe Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Entomology Commons Recommended Citation Tikhe, Chinmay Vijay, "Termite Gut Microbes as Tools and Targets for Termite Control" (2017). LSU Doctoral Dissertations. 4393. https://digitalcommons.lsu.edu/gradschool_dissertations/4393 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. TERMITE GUT MICROBES AS TOOLS AND TARGETS FOR TERMITE CONTROL A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Entomology By Chinmay Vijay Tikhe B.Sc. Microbiology, University of Pune, 2008 M.Sc. Microbiology, University of Pune, 2010 August 2017 Acknowledgments I would like thank my major advisor Dr. Claudia Husseneder for giving me the opportunity to conduct this research. Without her encouragement and support, this research wouldn’t have been possible. I will be always grateful for the trust and the intellectual freedom I received during this research. I would also like to thank Dr. Lane Foil, Dr. Chris Gissendanner and Dr. William Doerrler for serving on my PhD committee. I am really grateful for the valuable inputs I received from all of my committee members throughout this research. I highly appreciate Dr. Thomas Klei and Dr. Robert V. Rohli for serving as the Dean’s representative on my PhD committee. I am thankful to Dr. Mike Stout, Dr. James Ottea, and Dr. Timothy Schowalter and the Department of Entomology for funding my research. A conducive and stress-free environment created by all the wonderful lab mates, including Dr. Jong Seok Park, Mike Becker, Jennifer Dellate, Dawn Simms, Dr. Dee Colby, Hanah Shult, Thomas Martin, and Andrea Howells, helped me to stay focused and happy throughout this program. This research wouldn’t have been possible without the constant love and emotional support of my family. I cannot express my gratitude in words for my parents Sushama Tikhe and Vijay Tikhe. I am grateful to all the Tikhe and Bhalerao family members. I would like to express my gratitude to my wife Devika for playing innumerable important roles during the period of this research. I would like to thank all the following friends who stood by me in all the moments: Dr. Ashish Gupta, Dr. Dinesh Deochand, Dr. Sujeet Kumar, Dr. Ishan Joshi, and Dr. Sourav Chatterjee Special thanks to Nikhil Kulkarni, Shreyas Kulkarni, Pratik Shah, Sushant Gavhale, and Vikas Patil for their uninterrupted backing. ii Table of Contents Acknowledgments........................................................................................................................... ii Abstract ......................................................................................................................................... vii Chapter 1: Introduction ................................................................................................................... 1 1.1 Chemical methods used for termite control .......................................................................... 2 1.2 Biological methods used for termite control ......................................................................... 4 1.3 Termite gut protozoa as potential targets for termite control ................................................ 5 1.4 First research goal ................................................................................................................. 8 1.5 Termite gut bacteria as potential targets for termite control ................................................. 9 1.6 Second research goal ........................................................................................................... 11 1.7 References ........................................................................................................................... 12 Chapter 2: Isolation and assessment of gut bacteria from the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), for paratransgenesis research and application * .................................................................................................................................. 19 2.1 Introduction ......................................................................................................................... 19 2.2 Materials and methods ........................................................................................................ 21 2.2.1 Isolation of bacteria from the FST gut .......................................................................... 21 2.2.2 Sequencing of the 16S rRNA gene ............................................................................... 22 2.2.3 Minimum inhibitory concentration of Hecate and Ligand-Hecate ............................... 23 2.2.4 Transformation of Trabulsiella odontotermitis ............................................................ 24 2.2.5 Termite feeding bioassay .............................................................................................. 24 2.3 Results and discussion ......................................................................................................... 25 2.3.1 Bacteria identified from the termite gut ....................................................................... 25 2.3.2 Susceptibility to Hecate and ligand-Hecate .................................................................. 29 2.3.3 Termite feeding bioassay .............................................................................................. 32 2.4 References ........................................................................................................................... 35 Chapter 3: Assessment of genetically engineered Trabulsiella odontotermitis as a ‘Trojan Horse’ for paratransgenesis in termites * .................................................................................................. 40 3.1 Introduction ......................................................................................................................... 40 3.2 Materials and methods ........................................................................................................ 43 iii 3.2.1 Plasmid construction..................................................................................................... 43 3.2.2 Confirmation of the attTn7site in the Trabulsiella odontotermitis chromosome ......... 43 3.2.3 Preparation of electrocompetent cells and transformation of Trabulsiella odontotermitis ........................................................................................................................ 45 3.2.4 Confirmation of insertion of KanR cassette at attTn7site............................................. 46 3.2.5 Termite collection ......................................................................................................... 46 3.2.6 Feeding experiment ...................................................................................................... 46 3.2.7 Bacterial horizontal transfer ......................................................................................... 48 3.2.8 Consumption and mortality analysis ............................................................................ 49 3.2.9 Statistical analysis......................................................................................................... 50 3.4 Results and discussion ......................................................................................................... 50 3.4.1 Transformation with a constitutively expressed plasmid leads to strong but transient GFP expression in the termite gut ......................................................................................... 50 3.4.2 Trabulsiella odontotermitis engineered at chromosomal level at the attTn7 site......... 55 3.4.3 Chromosomally engineered T. odontotermitis is maintained in the termite gut for three weeks after ingestion ............................................................................................................. 57 3.4.4 Chromosomally engineered T. odontotermitis is horizontally transferred among nest mates ...................................................................................................................................... 59 3.4.5 Consumption and Mortality .......................................................................................... 62 3.5 References ........................................................................................................................... 64 Chapter 4: A pilot study to genetically engineering of Trabulsiella odontotermitis to express functional ligand-Hecate ............................................................................................................... 68 4.1 Introduction ......................................................................................................................... 68 4.2 Materials and methods .......................................................................................................