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Investigating the Molecular Basis of Resistance and Pyrethroid Selectivity at Acarine Sodium Channels

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Investigating the Molecular Basis of Resistance and Pyrethroid Selectivity at Acarine Sodium Channels ORBIT-OnlineRepository ofBirkbeckInstitutionalTheses Enabling Open Access to Birkbeck’s Research Degree output Investigating the molecular basis of resistance and pyrethroid selectivity at acarine sodium channels https://eprints.bbk.ac.uk/id/eprint/40252/ Version: Full Version Citation: Blockley, Alix Dawn (2017) Investigating the molecular basis of resistance and pyrethroid selectivity at acarine sodium channels. [The- sis] (Unpublished) c 2020 The Author(s) All material available through ORBIT is protected by intellectual property law, including copy- right law. Any use made of the contents should comply with the relevant law. Deposit Guide Contact: email INVESTIGATING THE MOLECULAR BASIS OF RESISTANCE AND PYRETHROID SELECTIVITY AT ACARINE SODIUM CHANNELS Alix Dawn Blockley MBiol (Hons) Thesis submitted to Birkbeck, University of London for the degree of Doctor of Philosophy September 2016 1 Declaration I declare that the work in this thesis is the candidates own. Signed: Alix Dawn Blockley 2 Acknowledgements Firstly, this work would not have been possible without the guidance of my wonderful supervisory team: Andreas Turberg, Bonnie Wallace, Emyr Davies, and in particular Lin Field and Martin Williamson. I thank you all for your advice and encouragement when it just would not work. Again. You have taught me how to supervise a student well. Thank you for always having an open door when I needed it. I particularly thank my “adoptive” supervisor, Ian Mellor at the University of Nottingham. Your advice, practical encouragement and supportive words, particularly when I was low on time and high on leak currents, made this work possible. I would also like to thank all those I have worked with at Rothamsted Research in the Insect Molecular Biology group and beyond. Thank you for making each day in the lab a good day. In particular, I thank Joel González-Cabrera, for his guidance at the start of my PhD, for providing the native Varroa destructor VGSC, and critically for sharing the frustrations of cloning arthropod VGSCs. I also thank Andrew Mead for his help with analysis of bioassay data and Rob King for bioinformatics support. To my lab group at Nottingham University: Rohit Patel, Neville Ngum, Abu Izuddin, Mahmoud Sherif, Maria Kor and David Richards. Thank you so much for the shared commiserations and congratulations, and for making me part of the team. In particular, I thank John Grzeskowiak for his friendship and for teaching me the basics of electrophysiology. I hope we can co-supervise a student or twenty! Lastly, I thank my husband James for his love and support, for keeping his phone on, washing my bike, and for not going to the Peaks without me. Your face. I dedicate this work to Sarah Bough, without whom I would never have started this PhD, wish you were here; and to my dad, who would have been a brilliant biologist. 3 Contents 1 Abbreviations Used .................................................................................................... 9 2 Abstract .................................................................................................................... 12 3 General Introduction ................................................................................................ 13 3.1 Acari .................................................................................................................. 13 3.2 Mites ................................................................................................................. 14 3.2.1 Mite Anatomy and Lifestyle ........................................................................... 14 3.2.2 Mite Damage and Mite-Borne Disease .......................................................... 14 3.3 Ticks ................................................................................................................... 20 3.3.1 Tick Anatomy and Lifestyle ............................................................................. 20 3.3.2 Tick Damage and Tick-Borne Disease ............................................................. 21 3.4 Acarine Control ................................................................................................. 25 3.4.1 Biological Acarine Control .............................................................................. 26 3.4.2 Vaccination ..................................................................................................... 28 3.4.3 RNAi Gene Silencing ....................................................................................... 31 3.4.4 Plant-Derived Chemical Acaricides ................................................................. 32 3.4.5 Synthetic Chemical Acaricides ........................................................................ 33 3.5 Pyrethroid Pesticides and the Arthropod VGSC ............................................... 35 3.5.1 Pyrethroid Discovery and Development ........................................................ 35 3.5.2 Expression of VGSCs ....................................................................................... 36 3.5.3 VGSC Structure and Function ......................................................................... 37 3.5.4 Pyrethroid Action at the Arthropod VGSC ...................................................... 41 3.5.5 The VGSC and Pyrethroid Resistance in Arthropods ...................................... 44 3.5.6 Pyrethroids as Acaricides ................................................................................ 45 3.6 Electrophysiological Methods to Study VGSC Activity ..................................... 47 3.6.1 Two Electrode Volt Clamp (TEVC) Electrophysiology ..................................... 47 3.6.2 Studying Pyrethroids and VGSCs Using TEVC Electrophysiology ................... 48 3.7 Aims and Objectives .......................................................................................... 49 4 General Experimental Methods ............................................................................... 50 4.1 Source Organisms ............................................................................................. 50 4.2 Agarose Gel Electrophoresis ............................................................................. 51 4 4.3 Extraction of Total RNA from Ticks and Mites .................................................. 51 4.4 Reverse Transcription of RNA ........................................................................... 53 4.5 Polymerase Chain Reaction (PCR) Protocols .................................................... 54 4.6 Primers .............................................................................................................. 54 4.7 Media and Antibiotics ....................................................................................... 54 4.8 Gel Purification of PCR Products ....................................................................... 55 4.9 Cloning of DNA for Sequence Analysis ............................................................. 56 4.10 Transformation of Plasmids into E. coli ............................................................ 57 4.11 Colony PCR ........................................................................................................ 58 4.12 Nucleic Acid Precipitation ................................................................................. 60 4.13 Extraction of Plasmid DNA from E. coli ............................................................. 60 4.14 Sequencing and Sequence Analysis .................................................................. 61 4.15 Site-Directed Mutagenesis of Cloned VGSC Sequences ................................... 61 4.16 Data Analysis ..................................................................................................... 63 5 Amplification and Sequencing of Acarine VGSCs ..................................................... 64 5.1 Chapter Introduction and Aims ........................................................................ 64 5.2 Chapter Specific Methods ................................................................................. 65 5.2.1 Primers for VGSC Amplification and Sequencing ........................................... 65 5.2.2 PCR Protocols .................................................................................................. 69 5.2.3 Rapid Amplification of cDNA Ends (RACE) ...................................................... 72 5.2.4 Transcriptomics .............................................................................................. 75 5.3 Results ............................................................................................................... 76 5.3.1 Comparing the Pyrethroid Binding Site of Insect and Acarine VGSCs ............ 76 5.3.2 Pyrethroid Resistance in R. microplus and R. sanguineus .............................. 81 5.4 Discussion.......................................................................................................... 86 6 Progress in Obtaining a Chimeric Arthropod VGSC ................................................. 90 6.1 Chapter Introduction and Aims ........................................................................ 90 6.2 Chapter Specific Methods ................................................................................. 90 6.2.1 para 13-5 Mutagenesis and Primer Design .................................................... 90 6.2.2 Amplification of the Domain-Spanning Region of the R. microplus VGSC ..... 94 6.2.3 Ligation Reactions to Create a Chimeric
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