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Studies of Captive Western Toad (Anaxyrus Boreas) Skin

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Studies of Captive Western Toad (Anaxyrus Boreas) Skin STUDIES OF CAPTIVE WESTERN TOAD (ANAXYRUS BOREAS) SKIN MICROBIOTA FOR POTENTIAL USE IN CHYTRIDIOMYCOSIS BIOCONTROL A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Biological Sciences By Marina E. De León 2017 SIGNATURE PAGE THESIS: STUDIES OF CAPTIVE WESTERN TOAD (ANAXYRUS BOREAS) SKIN MICROBIOTA FOR POTENTIAL USE IN CHYTRIDIOMYCOSIS BIOCONTROL AUTHOR: Marina E. De León DATE SUBMITTED: Summer 2017 Biological Sciences Department Dr. Wei-Jen Lin Thesis Committee Chair Biological Sciences Dr. Jill P. Adler-Moore Biological Sciences Dr. A. Kristopher Lappin Biological Sciences ii ACKNOWLEDGEMENTS I would like to extend my deepest appreciation for my advisor, Dr. Wei-Jen Lin, who chose to take a chance with me, and with this idea. Dr. Lin’s patience, kindness, and mentoring have meant everything to me. I am very grateful for my advisory committee, Dr. Jill Adler-Moore and Dr. Kristopher Lappin, who have given me intellectual contributions and unwavering support for this research. I also thank Jon Olson not just for his lab, but also his guidance and open-mindedness. Dr. Derek Sarovich provided so much more than his plasmids; he shared his knowledge as a true scientist, and I am indebted to him for always being available to answer all of my questions. Writing this thesis would not have been possible without the incredible support of my cat and turtle dad, Jimy Hu, Esq. I also need to thank my lab mates, who were always patient and taught me every microbiology technique I know- Ann Nasongkla, Liana Ab Samad, Justin Lee, Ashley Magin, and Danielle Valencia. My undergrad assistant and classmate Collin Knight was of great service during the MIC assays, and I thank him very much. I would also like to thank my oldest friend, and newest colleague, Heather M. Giddings- Provost for her writing suggestions. ii ABSTRACT Nearly 300 amphibian species are known to have been infected with Batrachochytrium dendrobatidis (Bd), the fungal pathogen that causes the severe skin infection chytridiomycosis. This infection is implicated in the extinction of over 100 species. Bacteria that produce antifungal compounds may give insight into possible treatments for this disease. Violacein, an antifungal metabolite naturally expressed by the soil bacterium Janthinobacterium lividum inhibits growth of the fungus. We explored the possibility of utilizing violacein as a biological control by attempting to introduce violacein genes into native Anaxyrus boreas skin bacterial isolates. Among 16 bacteria that were isolated from A. boreas, and identified using 16S rRNA sequencing, three Proteobacteria, and one in the FCB group, were used in transformation trials using a variety of plasmids. Three pPSX plasmid derivatives, each containing the violacein gene operon, were successfully transformed into laboratory Eschericia coli strains, but not into the wild-type skin bacteria that were induced for competence by chemicals, freeze/thaw, or electroporation. However, important discoveries were made about the ability of some wild-type bacteria to produce antifungal activities like J. lividum. In vitro inhibition assays against Bd strain JEL274 using native toad microflora and E. coli-violacein showed that a ubiquitous and highly abundant bacterium Chryseobacterium indologenes, of the FCB group, inhibited Bd significantly more than the E. coli-violacein transformants, and may have been involved with the captive toad’s ability to reduce or clear Bd infection. C. indologenes should be investigated further as a possible probiotic treatment against chytridiomycosis. iii TABLE OF CONTENTS Signature Page…………………………………………………………………………….ii Acknowledgements………………………………………………………………………iii Abstract…………………………………………………………………………………...iv List of Tables……………………………………………………………………………...x List of Figures…………………………………………………………………………….xi Chapter 1: Introduction……………………………………………………………………1 1.1. Amphibian Population Declines……………………………………………...1 1.2. Disease Transmission…………………………………………………………2 1.3. Disease Ecology………………………………………………………………3 1.4. Chytridiomycosis……………………………………………………………..5 1.5. Innate Immunity………………………………………………………………7 1.6. Adaptive Immunity…………………………………………………………...9 1.7. Genomic Analysis of Host Response………………………………………..10 1.8. Diversity of Amphibian Skin-Associated Bacteria……………………….…12 1.9. Anaxyrus boreas: Model Organism…………………………………………14 1.10. Bio-Augmentation as Treatment for Disease………………………………15 1.11. Janthinobacterium lividum………………………………………………...17 1.11.1 Structure and Function……………………………………………………17 1.11.2. Habitat……………………………………………………………………18 1.11.3. Metabolsim………………………………………………………………18 1.12. Violacein…………………………………………………………………...19 iv 1.13. Antifungal Bacteria………………………………………………………...22 1.13.1 Rana cascadae Skin Microbiota……...…………………………………..23 1.13.2 Anaxyrus boreas Skin Microbiota………………………………………..24 1.14. Limitations of Bioaugmentation…………………………………………...25 1.15. Limitations of Chytridiomycosis Drug Treatments………………………..26 1.16. Purpose of This Study……………………………………………………...28 Chapter 2: Materials and Methods……………………………………………………….31 2.1. Materials…………………………………………………………………….31 2.1.1. Microbial Culturing Media………………………………………..31 2.1.1.1. Tryptone…………………………………………… ……………31 2.1.1.2. Luria-Bertani (LB)………………………………………………………31 2.1.1.3. Tryptic Soy Broth (TSB)………………………………………………..31 2.1.1.4. Mueller Hinton (MH) …………………………………………………...31 2.1.1.5. Tryptone Gelatin hydrolysate Lactose (TGhL) …………………………32 2.1.2. Reagents…………………………………………………………………...32 2.1.2.1. Glycerol for Bacterial Storage and Wash……………………………….32 2.1.2.2. Electroporation Buffer…………………………………………………..32 2.1.2.3. Reagents for Chemical Competency…………………………………….33 2.1.2.4. Resazurin………………………………………………………………...33 2.1.2.5. Bd Cryoprotectant……………………………………………………….33 2.1.3. Antibiotics…………………………………………………………………33 2.1.3.1. Ampicillin……………………………………………………………….33 2.1.3.2. Trimethoprim …………………………………………………………...34 v 2.1.3.3. Amphotericin B………………………………………………………….34 2.1.4. Kits………………………………………………..……………………….34 2.1.4.1. Invitrogen Easy-DNA kit……………………………….……………….34 2.1.4.2. QIAquick PCR purification kit………………………………………….34 2.1.4.3. QIAprep Spin kit…………………………………..…………………….34 2.1.5. Molecular Biology Reagents and services………………...………...…….34 2.1.5.1. PCR Primers……………………………………………………….……34 2.1.5.2. Agarose Gel for Electrophoresis………………………………………...35 2.1.5.3. Sequencing………………………………………………………………35 2.2. Methods……………………………………………………………………………...35 2.2.1. Bacterial Collection…………………………………………………...…..35 2.2.2. Isolation of Bacteria……………………………………………………….36 2.2.3. Bacterial Subculture and Identification……………………………...……36 2.2.4. Bacterial Characterization Techniques……………………………………37 2.2.4.1. Gram Stain………………………………………………………………37 2.2.4.2. Capsule Stain……………………………………………………………37 2.2.4.3. Oxidase Test…………………………………………………………….37 2.2.4.4. Deep Agar Motility Test………………………………………..….……38 2.2.4.5. Wet Mount Motility Test………………………………………...……...38 2.2.4.6. Anaerobic Metabolism Test……………………………………………..38 2.2.5. Genomic DNA Extraction and 16S rRNA Sequencing…………………...38 2.2.6. Minimum Inhibitory Concentration (MIC) Analysis of Antibiotics Against Bacterial Isolates Using the Broth Microdilution Method……………………….39 2.2.6.1. Inoculum Preparation and Inoculation…………………………………..39 vi 2.2.6.2. Determining Minimal Inhibitory Concentration End Points……………41 2.2.7. Violacein-Bacteria Inhibition Assays……………………………………..41 2.2.8. Plasmids…………………………………………………………………...42 2.2.9. Transformation of Plasmids into Competent E. coli Cells………………..44 2.2.10. Preparation of Competent Cells: Growth to Mid Log Phase…………….45 2.2.11. Competency Induction Methods…………………………………...……45 2.2.11.1. Chemical Competency ………….………………………..……………45 2.2.11.2. Cell Wall Disruption Using the Freeze/Thaw Method………………...46 2.2.11.3. Competence for Electroporation……………………………………….46 2.2.12. Transformation Using Heat Shock Method and CaCl2 Competent Cells..47 2.2.13. Transformation Using Freeze/Thaw Method for Competent Cells……...47 2.2.14. Electroporation Transformation Trials…….…………………………….48 2.2.15. Verification of transformants by Restriction Digest……………….…….49 2.2.16. Growth and Maintenance of Bd………………………………………….49 2.2.16.1. Production of Zoospores……………………………………………….50 2.2.16.2. Harvesting Zoospores………………………………………………….50 2.2.16.3. Biosafety……………………………………………………………….50 2.2.16.4. Cryopreservation of Bd………………………………………………...50 2.2.17. Bd Detection using qPCR………………………………………………..51 2.2.18. Amphotericin B MIC Assays…………………………………….………51 2.2.19. Bd Inhibition Assays……………………………………………………..52 2.2.19.1. Scoring…………………………………………………………………52 2.2.20. Statistical Analysis of Data………………………………………………53 vii Chapter 3: Results………………………………………………………………………..54 3.1. Phenotypic Characterisation and Identification of Bacterial Isolates From CPP A. boreas Toads…………………………………………………………….54 3.2. Phylogenetic Relationships Among Bacterial Isolates from A. boreas……..57 3.3. Bd Inhibition Assays- Bacteria……………………………………………...58 3.4. Bd Inhibition Assays-Antifungal Drug Amphotericin B (AmB) ……..…….63 3.5. Bd Detection by qPCR………………………………………………………65 3.6. Antibiotic MIC Assays Against Bacterial Isolates…………...……………..65 3.7. Prescreening Transformation Candidates for Violacein Compatibility…..…69 3.8. Transformation of Violacein Plasmids to Bacterial Isolates………………...71 Chapter 4: Discussion……………………………………………………………………75 4.1. Overview…………………………………………………………………….75 4.2. Antibiotic Resistance in Environmental Bacteria…………………………...79 4.3. Is Transformation of Violacein
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