Michigan Technological University Digital Commons @ Michigan Tech Dissertations, Master's Theses and Master's Reports 2015 THE MECHANISMS OF α-AMANITIN RESISTANCE IN THE FRUIT FLY DROSOPHILA MELANOGASTER Chelsea L. Mitchell Michigan Technological University, [email protected] Copyright 2015 Chelsea L. Mitchell Recommended Citation Mitchell, Chelsea L., "THE MECHANISMS OF α-AMANITIN RESISTANCE IN THE FRUIT FLY DROSOPHILA MELANOGASTER", Open Access Master's Thesis, Michigan Technological University, 2015. https://doi.org/10.37099/mtu.dc.etdr/32 Follow this and additional works at: https://digitalcommons.mtu.edu/etdr Part of the Genetics Commons, and the Toxicology Commons THE MECHANISMS OF α-AMANITIN RESISTANCE IN THE FRUIT FLY DROSOPHILA MELANOGASTER By Chelsea L. Mitchell A THESIS Submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE In Biological Sciences MICHIGAN TECHNOLOGICAL UNIVERSITY 2015 © 2015 Chelsea L. Mitchell This thesis has been approved in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE in Biological Sciences. Department of Biological Sciences Thesis Advisor: Dr. Thomas Werner Committee Member: Dr. Rupali Datta Committee Member: Dr. Hairong Wei Department Chair: Dr. Chandrashekhar Joshi Table of Contents List of Figures .................................................................................................................... vi List of Tables .................................................................................................................... vii Preface.............................................................................................................................. viii Acknowledgements ............................................................................................................ ix Abstract ............................................................................................................................... x Introduction ......................................................................................................................... 1 Chapter 1 ............................................................................................................................. 6 1.1 Abstract ......................................................................................................................... 6 1.2 Introduction ................................................................................................................... 7 1.3 Results ........................................................................................................................... 9 1.3.1 After five decades without selective pressure, the three Asian fly stocks are still resistant to α-amanitin .......................................................................................... 9 1.3.2 α-Amanitin delays larva-to-adult development in a concentration-dependent manner ....................................................................................................................... 14 1.3.3 α-Amanitin reduces adult body size development in a concentration-dependent manner ....................................................................................................................... 16 1.3.4 α-Amanitin in the larval food increases egg-lay performance in adult females ....................................................................................................................... 18 1.3.5 α-Amanitin in larval food exerts a negative long-term effect on adult lifespan....................................................................................................................... 21 1.4 Discussion ................................................................................................................... 23 1.4.1 α-Amanitin resistance has no apparent fitness cost .......................................... 23 1.4.2 α-Amanitin causes developmental retardation phenotypes that resemble stress responses .................................................................................................................... 24 1.4.3 α-Amanitin increases female fecundity ............................................................ 26 1.4.4 Implications ...................................................................................................... 27 1.4.5 Limitations ........................................................................................................ 28 1.4.6 Future research ................................................................................................. 29 1.5 Conclusions ................................................................................................................. 30 1.6 Materials and Methods ................................................................................................ 31 iii 1.6.1 Fly stocks .......................................................................................................... 31 1.6.2 Generation of the isochromosomal lines .......................................................... 32 1.6.3 Dose-response studies of the fly stocks to α-amanitin ..................................... 32 1.6.4 Thorax measurements, fecundity, and longevity measurements ...................... 34 1.6.5 Statistical analyses ............................................................................................ 35 1.7 Acknowledgements ..................................................................................................... 35 1.8 Author Contributions .................................................................................................. 35 Chapter 2 ........................................................................................................................... 36 2.1 Abstract ....................................................................................................................... 36 2.2 Introduction ................................................................................................................. 37 2.3 Results ......................................................................................................................... 40 2.3.1 Experimental Design ........................................................................................ 40 2.3.2 Genes Encoding Cytochrome P450s, GSTs, and UGTs are Differentially Expressed in Ama-KTT/M/2 ..................................................................................... 43 2.3.3 Genes Encoding Cytochrome P450s and GSTs are Inducible by α-Amanitin ................................................................................................................. 46 2.3.4 Mdr Genes are Neither Constitutively Up-Regulated nor Inducible in Ama-KTT/M/2........................................................................................................... 48 2.3.5 Genome Enrichment Analysis Confirms 30-Year-Old QTL Mapping Data .... 50 2.3.6 Gene Ontology Enrichment Analysis Suggests Additional α-Amanitin Resistance Mechanisms ............................................................................................. 52 2.3.7 The Domain Enrichment Analysis Verifies the Gene Ontology Enrichment Analysis ..................................................................................................................... 57 2.3.8 The RT-qPCR Results Confirm the Microarray Data ...................................... 60 2.4 Discussion ................................................................................................................... 62 2.4.1 Several Mechanisms Seem to Confer α-Amanitin Resistance ......................... 62 2.4.2 Implications ...................................................................................................... 66 2.4.3 Limitations ........................................................................................................ 67 2.4.4 Future Studies ................................................................................................... 69 2.5 Conclusions ................................................................................................................. 71 2.6 Materials and Methods ................................................................................................ 71 2.6.1 Fly Stocks ......................................................................................................... 71 2.6.2 Generation of the isochromosome stock Ama-KTT/M/2 ................................. 72 iv 2.6.3 Dose-Response Studies of the Fly Stocks to α-Amanitin ................................. 72 2.6.4 Sample Preparation for the Microarray Analysis ............................................. 74 2.6.5 Affymetrix Array Target Preparation, Hybridization, and Scanning ............... 75 2.6.6 Microarray Data Normalization ........................................................................ 75 2.6.7 Genome Enrichment Analysis .......................................................................... 76 2.6.8 Identification of Differentially Expressed Genes (DEGs) ................................ 77 2.6.9 Gene Ontology Enrichment Analysis ............................................................... 78 2.6.10 Protein Domain Enrichment (PDE) Analysis ................................................. 78 2.6.11 RT-qPCR Validation of the Microarray Results ............................................ 79 2.7 Acknowledgments......................................................................................................