The Chemical Ecology of Host Plant Associated Speciation in the Pea Aphid (Acyrthosiphon Pisum) (Homoptera: Aphididae)
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The chemical ecology of host plant associated speciation in the pea aphid (Acyrthosiphon pisum) (Homoptera: Aphididae) By: David Peter Hopkins A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Sciences Departments of Animal and Plant Sciences October 2015 Table of Contents List of figures .......................................................................................................................... v List of tables .......................................................................................................................... viii Abbreviations .......................................................................................................................... ix Abstract .................................................................................................................................... x Acknowledgements ................................................................................................................. xi Chapter 1: General overview .................................................................................................. 1 1.1 Host race ecological divergence and speciation .......................................................... 2 1.2 A. pisum as a model system for studying ecological divergence of host-specialist races ..................................................................................................................................... 6 1.2.1 Evidence that plant chemistry is a source of divergent selection on A. pisum races ....................................................................................................................................... 10 1.2.2) Fabaceae as diverse chemical environments for A. pisum races ........................ 16 1.3 Overview of thesis ....................................................................................................... 19 Chapter 2: Profiling host plant acceptance and performance by two A. pisum races across multiple species of the plant genera Medicago and Trifolium. ........................................... 21 2.1 Chapter summary ........................................................................................................ 22 2.2 Introduction ................................................................................................................. 23 2.3 Methods ....................................................................................................................... 34 2.3.1 Plant material ........................................................................................................ 34 2.3.2 Aphid material ....................................................................................................... 35 2.3.3 EPG equipment and data capture ......................................................................... 35 2.2.4 Interpretation of raw EPG data ............................................................................. 37 2.3.5 Processing EPG waveform data and analysis of individual waveform measurements ............................................................................................................... 37 2.3.6 Interpretation of waveform measurements with multidimensional analysis ...... 38 2.3.7 Comparison of EPG acceptance profiles with aphid performance ....................... 40 2.4 Results and discussion ................................................................................................ 41 2.4.1 Correlation between clones within races ............................................................. 41 2.3.3 Multivariate analysis of acceptance using EPG data............................................. 50 2.3.4 Comparison of EPG profiles with fecundity and adult quality results. ................. 57 2.5 Conclusions .................................................................................................................. 64 i Chapter 3: Using aphid responses to multiple plant species to identify the chemical signatures behind host plant acceptance by two specialised races of pea aphid (Acyrthosiphon pisum) .......................................................................................................... 65 3.1 Chapter summary ........................................................................................................ 66 3.2 Introduction ................................................................................................................. 66 3.3 Methods ....................................................................................................................... 69 Methods used for aphid, plant culturing and EPG data collection are the same as for Chapter 2. These are outlined in brief below. ............................................................... 69 3.3.1 Aphid culture ......................................................................................................... 69 3.3.2 Plant culture .......................................................................................................... 70 3.3.3 Measuring aphid host preference using EPG ........................................................ 70 3.3.4 EPG profile of acceptance ..................................................................................... 71 3.3.5 Plant metabolomic profile .................................................................................... 72 3.3.6 Plant metabolome by aphid phenotype comparison using Random Forest regression ....................................................................................................................... 72 3.3.7 Putative mass identification and tandem mass spectrometry ............................. 73 3.4 Results ......................................................................................................................... 74 3.4.1 Analysis of EPG profiles ......................................................................................... 74 3.4.2 Random Forest models and identification of candidate masses .......................... 75 3.4.3 Analysis of L-phenylalanine and L-tyrosine pathways .......................................... 77 3.5 Discussion .................................................................................................................... 79 3.5.1 Integration of plant metabolomics with aphid genetic studies ............................ 85 3.6 Conclusion ................................................................................................................... 86 Chapter 4: The influence of host-plant pre-exposure to pea aphid (Acyrthosiphon pisum) aphids from two races upon aphid acceptance and key plant compounds. ....................... 87 4.1 Chapter Summary ........................................................................................................ 88 4.2 Introduction ................................................................................................................. 88 4.3 Methods ....................................................................................................................... 92 4.3.1 Plant culture .......................................................................................................... 92 4.3.2 Aphid culture ......................................................................................................... 92 4.3.3 Plant exposure to aphids ...................................................................................... 93 4.3.4 EPG profiling of plants exposed to aphid clones................................................... 93 4.3.5 Data processing and analysis of EPG recordings................................................... 94 4.3.6 Metabolic changes on plants exposed to two aphid clones ................................. 97 4.3.7 Targeted analysis of plant metabolic profiles ....................................................... 98 ii 4.4 Results ......................................................................................................................... 98 4.4.1 Number of probes ................................................................................................. 98 4.4.2 Phloem feeding behaviour as measured by waveform E2 ................................... 99 4.4. 4 Effect of exposure on targeted metabolites ...................................................... 107 4.5 Discussion .................................................................................................................. 109 4.5.1 Divergent acceptance towards constitutive, induced and subverted plant features. ....................................................................................................................... 109 4.5.2 Mechanism of divergence in A. pisum clone responses to plant chemistry ....... 113 4.5.3 Constitutive plant compounds and A. pisum acceptance ................................... 113 4.5.4 Evidence of induction and suppression by A. pisum ........................................... 115 4.5.5 Metabolic response of L-tyrosine to TP aphids .................................................. 119 4.6 Conclusion ................................................................................................................. 123 Chapter 5: Testing the direct effect of L-phenylalanine and L-tyrosine on A. pisum food preference using choice tests with artificial diets. ............................................................ 124 5 .1 Chapter