Transforming a Research Concept into Commercial Practice: Addressing the ‘Hurdles’ of Single-Species eDNA-based Detection Item Type Thesis or dissertation Authors Troth, Christopher Citation Troth, C.R. (2020) Transforming a Research Concept into Commercial Practice: Addressing the ‘Hurdles’ of Single-Species eDNA-based Detection. PhD Thesis. University of Derby. Publisher University of Derby Rights CC0 1.0 Universal Download date 26/09/2021 13:48:40 Item License http://creativecommons.org/publicdomain/zero/1.0/ Link to Item http://hdl.handle.net/10545/624574 UNIVERSITY OF DERBY Transforming a Research Concept into Commercial Practice: Addressing the ‘Hurdles’ of Single-Species eDNA-based Detection Christopher Robert Troth Doctor of Philosophy 2020 Contents Contents ........................................................................................................ 2 List of figures ................................................................................................. 6 List of tables .................................................................................................. 9 Abbreviations .............................................................................................. 11 Preface ........................................................................................................ 12 Abstract ....................................................................................................... 13 Acknowledgements...................................................................................... 15 Chapter 1: The ‘Hurdles’ associated with single-species eDNA-based detection methods ....................................................................................................... 17 1.1. Introduction .................................................................................................................... 17 1.1.1. Environmental DNA detection ........................................................................................................... 17 1.1.2. Development and history of the technique ....................................................................................... 18 1.1.3. Applied benefits of eDNA-based species detection methods ............................................................ 20 1.1.4. Present commercial use of eDNA-based detection methods within the UK and Europe .................. 22 1.2. Addressing the ‘hurdles’ associated with the commercial development and implementation of eDNA-based detection methods ......................................................................................... 23 1.2.1. Hurdle 1: Validation ........................................................................................................................... 25 1.2.2. Hurdle 2: Detection sensitivity ........................................................................................................... 28 1.2.3. Hurdle 3: Sample collection and preservation methodology ............................................................ 29 1.2.4. Hurdle 4: Sample site ......................................................................................................................... 30 1.2.5. Hurdle 5: Persistence and decay of eDNA ......................................................................................... 31 1.2.6. Hurdle 6: Environmental influences .................................................................................................. 31 1.2.7. Hurdle 7: Quantification .................................................................................................................... 33 1.2.8. Hurdle 8: Inhibition and contamination ............................................................................................. 34 1.2.9. Hurdle 9: Consistency and reliability ................................................................................................. 36 1.2.10. Hurdle 10: Commercial accessibility ................................................................................................ 39 1.3. The commercial potential of eDNA-based detection methods ........................................... 40 1.4. Conclusion ....................................................................................................................... 44 Chapter 2: Introduction to crayfish in the United Kingdom ........................... 45 2.1. Introduction .................................................................................................................... 45 2.1.1. White-clawed crayfish ....................................................................................................................... 45 2.1.2. Biology of white-clawed crayfish ....................................................................................................... 46 2.1.3. Life cycle of white-clawed crayfish .................................................................................................... 47 2.1.4. Populations in decline ........................................................................................................................ 50 2.1.5. Signal crayfish .................................................................................................................................... 52 2.1.6. Crayfish plague ................................................................................................................................... 54 2 2.1.7. Existing ecological survey methods for crayfish ................................................................................ 56 2.1.8. Crayfish and eDNA ............................................................................................................................. 57 2.1.9. White-clawed crayfish and eDNA ...................................................................................................... 60 2.2. Rationale ......................................................................................................................... 61 2.3. Thesis Aims ..................................................................................................................... 62 Chapter 3: The development and application of an eDNA-based methodology for the detection of white-clawed crayfish ................................................... 63 3.1. Abstract .......................................................................................................................... 63 3.2. Introduction .................................................................................................................... 64 3.3. Methodology ................................................................................................................... 66 3.3.1. Primer design and in-silico tests ........................................................................................................ 66 3.3.2. In-vitro validation ............................................................................................................................... 66 3.3.3. Limits of detection (LOD) and quantification (LOQ)........................................................................... 67 3.3.4. In-situ validation ................................................................................................................................ 67 3.3.5. Ex-situ comparison of eDNA sampling methodologies ...................................................................... 68 3.3.6. In-situ comparison of eDNA sampling methodologies ...................................................................... 70 3.3.7. Statistical analysis .............................................................................................................................. 71 3.4. Results ............................................................................................................................ 72 3.4.1. Assay development and in-silico and in-vitro validation ................................................................... 72 3.4.2. In-situ validation ................................................................................................................................ 72 3.4.3. Comparison of eDNA sampling methods ........................................................................................... 75 3.5. Discussion ....................................................................................................................... 78 3.5.1. Conclusion .......................................................................................................................................... 81 Chapter 4: Seasonality, DNA degradation and spatial heterogeneity as drivers of eDNA dynamics ........................................................................................ 83 4.1. Abstract .......................................................................................................................... 83 4.2. Introduction .................................................................................................................... 84 4.3. Methodology ................................................................................................................... 86 4.3.1. eDNA degradation experiment .......................................................................................................... 86 4.3.2. Seasonal dynamics in eDNA concentrations ...................................................................................... 87 4.3.3. Spatial and temporal in-situ variation...............................................................................................