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The School of Biological Sciences The University of Auckland New Zealand Identification and Analysis of Olfactory Receptors from the Light Brown Apple Moth, (Epiphyas postvittana) Doreen Shabana Begum March 2011 Supervisors: Richard D. Newcomb Andrew V. Kralicek David L. Christie A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biological Sciences, The University of Auckland, 2011.
Abstract Abstract Olfaction or the sense of smell is one of the major modes of communication in moths, playing an integral part in the moths‟ ability to locate mates for reproduction, location of host plants and oviposition sites. Some of the key players of the moths‟ olfactory system include general odorant binding proteins and olfactory receptors (OR). The aim of this study was to investigate the molecular mechanisms of olfaction in the light brown apple moth, Epiphyas postvittana. These data will contribute to the development of new pest control strategies for the moth and the development of olfactory biosensors. E. postvittana OR1 (EpOR1) is closely related to the pheromone receptors (PR) of other lepidopterans, but is expressed at similar levels in male and female antennae. Functional analysis in Sf9 cells demonstrated that it binds important plant semiochemicals but not pheromone. EpOR1 is sensitive to methyl salicylate, recognising this odorant to a low concentration of 10 -15 M. EpOR1 also binds a range of terpenes which showed Hill slopes ranging from 0.33–3.2. E. postvittana GOBP2 (EpGOBP2) was expressed in bacteria and purified to homogeneity. Binding analysis revealed it was able to bind octanol, α-farnesene, methyl salicylate, nerol, eucalyptol, geranyl acetate and pentyl acetate, but not citral, geranial and geraniol. EpGOBP2 was able to replace DMSO as a solubilising agent for methyl salicylate and geranyl acetate in a functional assay of EpOR1 in Sf9 cells. The sensitivity of EpOR1 for these two ligands in vitro was enhanced in the presence of EpGOBP2 [EC50 of EpOR1 with methyl salicylate in DMSO is (1.8 ± 0.9) x 10 - 12 M and EC50 of EpOR1 with methyl salicylate in the presence on EpGOBP2 is (1.19 ± 0.82) x 10 -13 M], suggesting that EpGOBP2 might not only be acting as a solubilising agent but also as an activated ligand. Forty-nine new ORs were identified by deep transcriptomic sequencing and light coverage genome scanning of E. postvittana bringing the total number of ORs identified to date for this species to 52. Phylogenetic analysis revealed that associated with lineage, 24 ORs had one-to-one orthologs with ORs from the silkworm, Bombyx mori, while 28 E. postvittana ORs were expansions compared with B. mori. EpOR1 i
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Page 5 and 6: Acknowledgements Acknowledgements I
Page 7 and 8: Contents v Contents Abstract……
Page 9 and 10: Contents 4.2.5 Degenerate PCR .....
Page 11 and 12: List of Figures List of Figures Fig
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Page 15 and 16: List of Abbreviations gDNA Genomic
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Functional characterisation of Epip
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3.1 Introduction 3 The roles of Epi
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4.1 Introduction 4 Identification o
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Identification of putative odorant
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Microarray OR 454 Transcriptome OR
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5.1 Introduction 5 Concluding Discu
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Concluding Discussion 127 VOBA sett
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Concluding Discussion 129 redundant
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Concluding Discussion 131 against t
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Concluding Discussion 133 sequencin
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Appendix B 135 Buffered TB (Sambroo
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Appendix C 137 C. Appendix C - Clus
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Appendix D 139 D. Appendix D - Solu
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Appendix E 141 I II
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Appendix E 143 V
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Appendix E 145 VII continued Figure
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References 147 Ansebo, L., Ignell,
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References 149 Butenandt, A., Beckm
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References 151 Fedurco, M., Romieu,
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References 153 Große-Wilde, E., St
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References 155 Hrdy, I., F. Kuldova
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References 157 Kaissling, K. E. (19
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References 159 Krieger, J., Große-
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References 161 Lopez-Gomez, R. and
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References 163 Miura, N., Nakagawa,
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References 165 Pell, J. K., Macaula
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References 167 Røstelien, T., Stra
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References 169 Stowers, L. and Loga
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References 171 Turner, C. T., Davy,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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