INVESTIGATING THE MOLECULAR MECHANISMS OF INSECTICIDE RESISTANCE IN THE TOMATO LEAF MINER, TUTA ABSOLUTA MADELEINE BERGER Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy SEPTEMBER 2015 Abstract Tuta absoluta is an economically significant pest of tomatoes, which has undergone a rapid expansion in its range during the past six years. One of the main means of controlling this pest is through the use of chemical insecticides including pyrethroids and spinosad. However, their intensive use has led to the development of resistance. The aim of this PhD was to understand the mechanisms underlying resistance to pyrethroids and spinosad. The target site of pyrethroids, the sodium channel, was cloned and three known knockdown resistance mutations, L1014F, M918T and T929I were found. High-throughput diagnostic assays were developed and the prevalence of the three mutations was then assessed. All three mutations were found at high frequencies in populations across the range of T. absoluta. Additionally, a fourth novel mutation L925M was found in 14% of samples. Therefore, pyrethroids are unlikely to be effective at controlling T. absoluta. Bioassays were conducted to determine the sensitivity of five populations of T. absoluta to spinosad. One population, from an area where control failure using spinosad was reported in 2012, exhibited a high level of resistance after selection in the laboratory with spinosad. Synergist bioassays did not show enhanced activity/expression of P450s and esterases. The transcriptome of T. absoluta was sequenced and used, in combination with degenerate PCR, to identify the target site of spinosad, the nicotinic acetylcholine receptor (nAChR) α6 subunit. Analysis of Taα6 revealed that two mutually exclusive exons (3a and 3b) that encode loop D of the ligand binding domain are both absent in all transcripts from the selected strain. Additionally, QPCR showed that α6 is down regulated in both larvae and adults of the selected strain. Taken together this study has provided new data on the molecular basis of resistance of T. absoluta to pyrethroids and spinosad. Acknowledgements I would like to thank my supervisors Chris Bass, Lin Field and Martin Williamson at Rothamsted Research and Ian Mellor and Ian Duce at the University of Nottingham for all of their help throughout my PhD. I would also like to thank everyone in the insect molecular biology lab including Alix Blockley, Asa Nordgren, Emma Randall, Katherine Beadle, Will Garrood, Mirel Puinean, Christoph Zimmer, Sophia Iqbal, Mark Mallott, Emyr Davies, Sonia Rodriguez-Vargas, Joel Gonzalez and Bartek Troczka. I would like to thank Keywan Hassani-Pak and David Hughes for teaching me bioinformatics analysis, Suzanne Clark for statistics advice and Chris Jones for advice on DEseq2. I would like to thank Kevin Gorman and Khalid Haddi for teaching me how to rear T. absoluta and conduct bioassays. I thank Steve Harvey for growing my tomato plants and Mark Mallott for help building my selection chamber. I would like to thank the insectary staff including Liz Iger, Linda Oliphant, Di Cox and Nigel Watts. I would like to thank Ian Denholm for his help setting up collaboration between Rothamsted Research and Pernambuco Federal Rural University (UFRPE) in Brazil. I am very grateful to Herbert Siqueira, Wellington Marques de Silva, Agna Rita dos Santos Rodrigues and everyone in the insect toxicology laboratory at UFPRE for hosting me during my visit. Thank you to the European Union for funding this visit under the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ REA, grant agreement PIRSES-GA-2012 – 318246. Finally, I would like to thank BBSRC for funding my PhD. Table of Contents 1. General Introduction 1.1 Food security 1 1.2 Insect pests 2 1.2.1 Tuta absoluta 2 1.3 Control of insect pests 7 1.3.1 Biological control 7 1.3.1.1 Biological control of T. absoluta 8 1.3.2 Chemical control 9 1.3.2.1 Chemical control of T. absoluta 9 1.3.2.2 Pyrethroids 11 1.3.2.3 Spinosyns 13 1.4 Regulation of Splicing 17 1.5 Mechanisms of insecticide resistance 19 1.5.1 Reduced penetration 19 1.5.2 Metabolic Resistance 20 1.5.3 Target-site resistance 21 1.6 Resistance management 24 1.7 Objectives 24 2. General materials and methods 2.1 T. absoluta populations 26 2.1.1. Live insect populations 26 2.1.2. Preserved insect material 26 2.2 DNA extraction 27 2.2.1. DNAzol® (Life Technologies, USA) 27 2.2.2. DNeasy® Plant Mini Kit (Qiagen, Germany) 28 2.3 RNA extraction and cDNA synthesis 28 2.4 Polymerase Chain Reactions (PCRs) 30 2.4.1 Standard PCR 30 2.4.2 Long PCR 30 2.5 Purification of PCR Products 31 2.6 Cloning of PCR fragments 31 2.7 Sequencing of PCR fragments and plasmids 32 2.8 Rapid Amplification of cDNA ends (RACE) 33 2.9 Genome Walking 33 2.10 Quantitative PCR (qPCR) 34 3. Resistance to pyrethroids 3.1 Introduction 37 3.2 Specific Methods 38 3.2.1 Pyrethroid Bioassays 38 3.2.2 Cloning and sequencing of regions encoding domain II of the T. 39 absoluta sodium channel 3.2.3 TaqMan® PCR 40 3.3 Results and Discussion 41 3.3.1 Susceptibility of five laboratory populations of T. absoluta to 41 pyrethroids 3.3.2 Cloning and sequencing of regions of the T. absoluta sodium 42 channel gene 3.3.3 TaqMan assays to determine frequency of L1014F, M918T and 45 T929I in T. absoluta populations 3.3.4 Geographical distribution of the three pyrethroid-resistance 50 mutations in T. absoluta 3.3.5 Detection of kdr/skdr in field populations of T. absoluta from 51 Brazil 3.3.6 Detection of a fourth novel mutation in T. absoluta. 52 3.4 Conclusions 55 4. Bioassays to determine sensitivity of T. absoluta to spinosad 4.1 Introduction 57 4.2 Specific Methods 58 4.2.1 Insect material 58 4.2.2 Selection of the Spin-Parent population to give the SpinSel 58 strain 4.2.3 Bioassays of T. absoluta Larvae 59 4.2.3.1 Leaf-dip bioassays 59 4.2.3.2 Synergist Assays 60 4.2.4 Bioassays of T. absoluta Adults 60 4.2.4.1 Leaf-dip bioassays 60 4.2.4.2 Topical bioassays 61 4.2.4.3 Feeding bioassays 61 4.2.5 Statistical analysis 61 4.3 Results and Discussion 62 4.3.1 Susceptibility of T. absoluta populations 62 4.3.2 Initial susceptibility of the T. absoluta Spin-Parent population 63 4.3.3 Selection of the Spin-Parent population to give the SpinSel 63 strain 4.3.4 Synergist assays 65 4.3.5 Adult bioassays 66 4.4 Conclusions 67 5. Generation of T. absoluta transcriptome 5.1 Introduction 70 5.2 Specific Methods 70 5.2.1 454 sequencing 70 5.2.2 Illumina sequencing 71 5.2.2.1 Sequencing of T. absoluta strain TA1 71 5.2.2.2 Sequencing of T. absoluta strains Spin and SpinSel 71 5.2.3 Newbler de-novo assembly 71 5.2.4 Trinity de-novo assembly 72 5.2.5 Annotation 72 5.3 Results and Discussion 73 5.3.1 Transcriptome assemblies 73 5.3.2 Blast analysis of transcriptomes 77 5.3.3 Transcripts encoding cytochrome P450s and insecticide target 81 sites 5.4 Conclusions 87 6. Analysis of the nAChR α6 subunit 6.1 Introduction 88 6.2 Specific Methods 89 6.2.1 Cloning and sequencing of the nAChR α6 subunit 89 6.2.2 Sequencing of the genomic T. absoluta nAChR α6 subunit 90 6.2.3 Analysis of differentially expressed transcripts 91 6.2.4 Comparison of nAChR α6 subunits from different life stages of 93 T. absoluta 6.3 Results and Discussion 93 6.3.1 Cloning of the nAChR α6 subunit from T. absoluta 93 6.3.2 Comparison of Spin and SpinSel nAChR α6 subunit cDNA 97 sequences 6.3.3 Sequencing of nAChR α6 subunit gDNA sequences from 99 SpinSel 6.3.4 RNA-seq analysis of Spin and SpinSel 104 6.3.4.1 Expression of regulators of splicing 106 6.3.5 Comparison of nAChR α6 subunit of T. absoluta in different 110 life stages 6.3.6 Relative expression of nAChR α6 subunit of T. absoluta 111 6.4 Conclusions 114 116 7. General Discussion 7.1 Pyrethroid resistance in T. absoluta 116 7.2 Spinosad resisance in T. absoluta 119 7.3 Implications for resistance management of T. absoluta 123 7.4 Future work 126 References 127 Appendix 1. Insect Biochemistry and Molecular Biology (2012) 42, 141 506-513. Appendix 2. Pesticide Biochemistry and Physiology (2015) 122, 8- 149 14. Appendix 3A. Contigs with hits to cytochrome p450s, 454 156 sequencing (Assembly 1) Appendix 3B. Contigs with hits to cytochrome p450s, Illumina 159 sequencing (Assembly 2) Appendix 4. Amino acid sequences of insecticide target sites 168 assembled from Illumina transcriptomes Appendix 5. Alignment of Spin, SpinSel and TA4 genomic DNA 177 sequence Appendix 6A. Differentially expressed transcripts in Assembly 5 180 Appendix 6B. Differentially expressed transcripts in Assembly 6 187 List of Tables Table 1.1. Insecticide classes registered for use against T. absoluta 11 Table 1.2. Number of potential insecticide detoxification genes in 21 selected insects Table 2.1. Origin of live populations of T. absoluta 26 Table 2.2. Modifications to DNAzol® protocol 28 Table 2.3. Housekeeping gene primers 36 Table 3.1. Primers used for amplification of the Tuta absoluta para- 40 type sodium channel and TaqMan assays Table 3.2.