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Detection and Quantification of an Ecologically Important Marine

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Detection and Quantification of an Ecologically Important Marine by Robyn Buchwald Submitted in partial fulfillment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia August 2016 © Copyright by Robyn Buchwald, 2016 TABLE OF CONTENTS List of Tables ................................................................................................................... vi List of Figures ................................................................................................................ viii Abstract ............................................................................................................................ xi List of Abbreviations and Symbols Used ..................................................................... xii Acknowledgements ....................................................................................................... xiv Chapter 1. Introduction .................................................................................................. 1 Chapter 2. Development of a Species-Specific PCR/qPCR Assay for Detection and Quantification of Paramoeba invadens ................................................................... 4 2.1. Introduction ............................................................................................................. 4 2.2. Materials and Methods ............................................................................................ 8 2.2.1. Sequencing P. invadens and Parasome ITS Regions ....................................... 8 2.2.2. P. invadens-Specific PCR design .................................................................... 9 2.2.2.1. Primer Design ........................................................................................... 9 2.2.2.2. Primer Specificity Testing ........................................................................ 9 2.2.2.3. Primer Sensitivity Testing ....................................................................... 10 2.2.2.4. PCR Optimization of Selected Primer Sets ............................................ 11 2.2.3. Host Tissue and Environmental Sample Processing ...................................... 11 2.2.3.1. Sample Collection ................................................................................... 11 2.2.3.2. Culturing P. invadens .............................................................................. 12 2.2.3.3. Culturing versus Molecular Identification of Infection with P. invadens, and Radial Nerve DNA Extraction ...................................................... 13 2.2.3.4. Sediment DNA Extraction ...................................................................... 13 2.2.3.5. Seawater DNA Extraction ....................................................................... 14 ii 2.2.3.6. PCR Reaction Conditions for the Detection of P. invadens in Radial Nerve, Sediment and Seawater DNA Samples ......................................... 14 2.2.3.7. Confirmation of Amplicon Identity from Environmental DNA Samples ................................................................................................................ 15 2.2.4. Inferring PCR Assay Sensitivity for Environmental Samples ....................... 15 2.2.5. Quantitative Real-Time PCR with SYBR Green ........................................... 17 2.2.5.1. Quantitative PCR Reaction Conditions and Optimization ...................... 17 2.2.5.2. Calibration Curves for Quantification of P. invadens ............................ 17 2.2.5.3. Genomic Copy Number of 18S rRNA Gene in P. invadens ................... 18 2.2.5.4. Reliability of Cell Number Estimates Obtained using qPCR ................. 18 2.3. Results ................................................................................................................... 19 2.3.1. Sequencing P. invadens and Parasome ITS Regions ..................................... 19 2.3.2. P. invadens-Specific PCR: Design and Optimization .................................... 20 2.3.2.1. Primer Design ......................................................................................... 20 2.3.2.2. Testing Primer Specificity ...................................................................... 21 2.3.2.3. Testing Primer Sensitivity ....................................................................... 23 2.3.2.4. PCR Optimization of Selected Primer Sets ............................................ 24 2.3.3. Host Tissue and Environmental Sample Processing ...................................... 25 2.3.3.1. Culturing versus Molecular Identification of P. invadens Infection ....... 25 2.3.3.2. Confirmation of Amplicon Identity from Environmental DNA Samples ................................................................................................................ 27 2.3.4. Inferring PCR Assay Sensitivity for Environmental Samples ....................... 27 2.3.4.1. Radial Nerve DNA .................................................................................. 27 2.3.4.2. Sediment DNA ........................................................................................ 30 2.3.4.3. Seawater DNA ........................................................................................ 31 2.3.5. Quantitative Real-Time PCR ......................................................................... 33 iii 2.3.5.1. qPCR Optimization ................................................................................. 33 2.3.5.2. Estimation of Copy Number in P. invadens Cells .................................. 36 2.3.5.3. Reliability of Cell Number Estimates Obtained using qPCR ................. 37 2.4. Discussion ............................................................................................................. 37 2.4.1. Contributions of the Present Study ................................................................ 37 2.4.2. Selection of DNA Extraction Techniques ...................................................... 38 2.4.3. PCR Sensitivity in Radial Nerve, Sediment, and Seawater DNA ................. 38 2.4.4. Reliability of Cell Estimates Obtained using qPCR ...................................... 40 2.4.5. qPCR Inhibition and Optimization ................................................................ 40 2.4.6. Limitations of qPCR using SYBR Green and Recommendations ................. 41 2.4.7. Suggestions for Future Research ................................................................... 42 Chapter 3. Detection and Quantification of Paramoeba invadens Along the Atlantic Coast of Nova Scotia ....................................................................................... 44 3.1. Introduction ........................................................................................................... 44 3.2. Materials and Methods .......................................................................................... 46 3.2.1. Sampling Design ............................................................................................ 46 3.2.2. Sample Analysis ............................................................................................. 48 3.2.2.1. Radial Nerve Plating and Radial Nerve, Sediment, and Seawater DNA Extraction ................................................................................................... 48 3.2.2.2. Detection of P. invadens by PCR ........................................................... 49 3.2.2.3. Quantification of P. invadens in Radial Nerve Tissue and Seawater ..... 49 3.3. Results ................................................................................................................... 50 3.3.1. Detection of P. invadens in Sea Urchins, Sediments and Seawater using PCR ................................................................................................................ 50 3.3.2. Quantification of P. invadens in Sea Urchins and Seawater using qPCR ........................................................................................................................ 54 iv 3.3.2.1. Sea Urchins ............................................................................................. 54 3.3.2.2. Seawater .................................................................................................. 55 3.4. Discussion ............................................................................................................. 59 3.4.1. Detection of Paramoeba invadens in Sea Urchins and Sediment ................. 59 3.4.2. Detection of Paramoeba invadens in Seawater ............................................. 62 3.4.3. Conclusions and Prospects for Future Research ............................................ 63 Chapter 4. Discussion .................................................................................................... 65 4.1. The Utility of Molecular Tools for Understanding the Epizootiology of Paramoeba invadens .................................................................................................... 65 4.2. Perspectives on the Mechanisms of Introduction of Paramoeba invadens to Nova Scotia .................................................................................................................. 66 Appendix A: Supplementary Materials ....................................................................... 68 Bibliography ................................................................................................................... 82 v LIST OF TABLES Table 2.1. Paramoeba invadens isolates used to sequence
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