Alternative methods for assessing habitat quality in freshwater systems Item Type Thesis or dissertation Authors Mauvisseau, Quentin Citation Mauvisseau, Q 2020, 'Alternative methods for assessing habitat quality in freshwater systems', PhD thesis, University of Derby, Derby Publisher University of Derby Download date 28/09/2021 22:40:52 Link to Item http://hdl.handle.net/10545/625067 University of Derby Alternative methods for assessing habitat quality in freshwater systems Quentin Mauvisseau Doctor of Philosophy 2020 THIS PAGE HAS BEEN INTENTIONALLY LEFT BLANK 2 CONTENTS Figures............................................................................................................................................. 7 Tables .............................................................................................................................................. 9 Appendices .................................................................................................................................... 11 Abbreviations ................................................................................................................................ 12 Preface........................................................................................................................................... 14 Abstract ......................................................................................................................................... 17 Acknowledgment .......................................................................................................................... 19 Chapter 1: Introduction ................................................................................................................. 21 1.1 Current state ........................................................................................................................ 21 1.2 Conventional monitoring methods ...................................................................................... 22 1.3 eDNA-based detection as an alternative monitoring method? ............................................ 23 1.4 Aims of the thesis ................................................................................................................ 26 Chapter 2: Influence of accuracy, repeatability and detection probability in the reliability of species-specific eDNA based approaches ..................................................................................... 29 2.1 Abstract ............................................................................................................................... 29 2.2 Introduction ......................................................................................................................... 30 2.3 Materials and methods ........................................................................................................ 34 2.3.1 Study species and system ............................................................................................. 34 2.3.2 Sampling and PCR protocols ........................................................................................ 35 2.3.3 Statistical analysis......................................................................................................... 38 2.4 Results ................................................................................................................................. 39 2.5 Discussion ........................................................................................................................... 45 Chapter 3: eDNA based monitoring: advancement in management and conservation of critically endangered killifish species .......................................................................................................... 49 3.1 Abstract ............................................................................................................................... 49 3.2 Introduction ......................................................................................................................... 50 3.3 Methods ............................................................................................................................... 52 3.3.1 Assay development ....................................................................................................... 52 3 3.3.2 eDNA extraction ........................................................................................................... 53 3.3.3 PCR and qPCR ............................................................................................................. 54 3.3.4 qPCR analysis ............................................................................................................... 54 3.3.5 Ex-situ testing of the eDNA assays .............................................................................. 55 3.3.6 In-situ trial 1 (A) for V. robertae and V. letourneuxi ................................................... 56 3.3.7 In-situ trial 2 (B) for all three fish species .................................................................... 58 3.3.8 Statistical analysis......................................................................................................... 60 3.4 Results ................................................................................................................................. 60 3.5 Discussion ........................................................................................................................... 65 Chapter 4: Improving detection capabilities of a critically endangered freshwater invertebrate with environmental DNA using digital droplet PCR ............................................................................ 70 4.1 Abstract ............................................................................................................................... 70 4.2 Introduction ......................................................................................................................... 71 4.3 Methods ............................................................................................................................... 72 4.3.1 Primers and probe design ............................................................................................. 72 4.3.2 eDNA samples .............................................................................................................. 73 4.3.3 DNA extraction............................................................................................................. 75 4.3.4 PCR ............................................................................................................................... 75 4.3.5 qPCR ............................................................................................................................. 75 4.3.6 ddPCR ........................................................................................................................... 76 4.3.7 Estimation of the LOD and LOQ ................................................................................. 77 4.3.8 Kick-sampling .............................................................................................................. 77 4.3.9 Statistical analysis......................................................................................................... 77 4.4 Results ................................................................................................................................. 78 4.4.1 Specificity and validation of eDNA assay using PCR, qPCR and ddPCR................... 78 4.4.2 Kick sampling assessment ............................................................................................ 79 4.4.3 Comparison of qPCR versus ddPCR analyses ............................................................. 79 4.5 Discussion ........................................................................................................................... 82 Chapter 5: The development of an eDNA based detection method for the invasive shrimp Dikerogammarus haemobaphes.................................................................................................... 86 5.1 Abstract ............................................................................................................................... 86 4 5.2 Introduction ......................................................................................................................... 86 5.3 Methods ............................................................................................................................... 88 5.3.1 Primers and probe design ............................................................................................. 88 5.3.2 eDNA samples .............................................................................................................. 90 5.3.3 DNA extraction............................................................................................................. 90 5.3.4 PCR and qPCR amplification ....................................................................................... 92 5.3.5 Statistical analysis......................................................................................................... 93 5.4 Results ................................................................................................................................. 93 5.5 Discussion 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