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Concluding Discussion 130 components or test odorants may be toxic to the OR being tested and an understanding of the roles of other olfactory players in these in vitro systems may help overcome these limitations. Functional studies of EpOR6, 30, 33 and 34 will enable the determination of their roles in E. postvittana olfaction. The phylogenetic relatedness of EpOR6 to the pheromone receptors from other moths confers it as a PR candidate, however, tissue expression analysis and functional characterisation of this candidate OR will deduce its role in E. postvittana olfaction. The relatively high expression of EpOR30, 33 and 34 in male moth antennae, as shown by qRT-PCR is suggestive of a plausible role in pheromone recognition of these ORs. In situ hybridisation of these candidates can be used to test if they are likely PRs. ORs that are expressed at the base of pheromone sensitive trichoid sensilla of male moths will likely be PRs. Functional analysis by cloning, expression and characterisation in an assay system against E. postvittana pheromone components will confirm their binding partners. The functional characterisation of PRs from moths such as B. mori and H. virescens has indicated the presence of multiple PRs within each species which are tuned to recognising one or more of the sex pheromone components. It is tempting to postulate the existence of multiple PRs in E. postvittana also and one or more of the four candidates could be the PRs of E. postvittana, however, further functional analysis will have to be done to test the roles of these ORs. If any of these four candidates are found to be the PR for E. postvittana, then further strategies can be developed in dealing with this agricultural pest. Mimics that can act as irreversible blocking agents for the binding site of the PR could be developed. A mimic that will not be cleared from the receptor after signal attenuation would be ideal (native pheromones are cleared from the receptor hence large amounts are needed in mating disruption studies, rendering this pest control method expensive). Its release into E. postvittana infested areas could see a reduction in the number of successful matings as the mimic will flood the PR and confuse the male moth. Another strategy would be to genetically modify the PR itself so that it no longer recognises the pheromone components. This can be done by completely shutting off the PR by RNAi silencing. Small interfering RNA or double-stranded RNA (dsRNA) could be sprayed on host plants, or genetically modified plants expressing dsRNA
Concluding Discussion 131 against the receptor could be introduced (pertaining ethical approvals). Larvae feeding on these plants will have their PRs turned off or modified, hence will be unable to recognise sex pheromones and the number of successful matings will decrease. Turner et al. (2006) fed dsRNA to larvae and demonstrated the effective knockdown of genes expressed in the adult antennae (Turner et al., 2006). This method will however require the GM plants, siRNA and dsRNA sprays to undergo various tests to render them safe for the environment and humans. During the course of this project, 49 putative ORs were identified, however, the full length sequences of 48 of these still need to be completed. Towards the end of this study, six more lanes of Solexa genome sequences was obtained. However the data was not available on time to include in this thesis. This new data combined with the previous genomic sequence dataset will hopefully give longer scaffolds with longer, if not full length coding regions of the putative ORs. Further novel ORs could also be identified from the new data, thereby increasing the receptor repertoire of E. postvittana. Functional characterisation of these full length ORs can be done to identify male and female specific ORs and their binding components. A detailed functional characterisation of these ORs will facilitate the decoding of all these ORs, and an odorant–OR map can be built for E. postvittana. Such a study will provide data that can be used as a basis to create a computer model to predict binding partners for ORs in related moths. A computer model that can for example narrow down on „x‟ number of candidate odorants for testing an OR with can be used as a first screening towards characterising novel ORs. This will save the researcher physically testing tens to hundreds of compounds per receptor to identify its binding components. The only lepidopteran for which a draft genome sequence is publically available to date is the silkmoth B. mori. This is a domesticated moth which is dependent on humans for survival and reproduction. Currently it is used as the model for Lepidoptera studies, including tortricid moths however, the availability of genome data for an insect pest will be invaluable to Tortricidae genomic biology, especially in understanding the underlying molecular mechanisms of insect pests and combating the disruptive members of this family. Keeping this in mind, the genome sequencing of E. postvittana has been initiated. E. postvittana is perhaps one of the most disruptive members of this family, in that it is highly polyphagous and can survive in
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
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Contents 4.2.5 Degenerate PCR .....
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List of Figures List of Figures Fig
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List of Tables List of Tables Table
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List of Abbreviations gDNA Genomic
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1.1 General overview 1 General Intr
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General Introduction 3 moth in loca
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General Introduction 5 Figure 1.1:
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General Introduction 7 1.5 Componen
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General Introduction 9 OBPs have al
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General Introduction 11 A model has
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General Introduction 15 Tanaka et a
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General Introduction 17 Table 1.1:
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General Introduction 21 et al., 200
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General Introduction 23 the attract
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General Introduction 25 leaves and
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General Introduction 29 Expression
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General Introduction 31 BmOr2 HvOr2
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General Introduction 33 for lepidop
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Functional characterisation of Epip
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3.1 Introduction 3 The roles of Epi
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The roles of Epiphyas postvittana G
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Page 151 and 152: Appendix B 135 Buffered TB (Sambroo
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Page 157 and 158: Appendix E 141 I II
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Page 161 and 162: Appendix E 145 VII continued Figure
Page 163 and 164: References 147 Ansebo, L., Ignell,
Page 165 and 166: References 149 Butenandt, A., Beckm
Page 167 and 168: References 151 Fedurco, M., Romieu,
Page 169 and 170: References 153 Große-Wilde, E., St
Page 171 and 172: References 155 Hrdy, I., F. Kuldova
Page 173 and 174: References 157 Kaissling, K. E. (19
Page 175 and 176: References 159 Krieger, J., Große-
Page 177 and 178: References 161 Lopez-Gomez, R. and
Page 179 and 180: References 163 Miura, N., Nakagawa,
Page 181 and 182: References 165 Pell, J. K., Macaula
Page 183 and 184: References 167 Røstelien, T., Stra
Page 185 and 186: References 169 Stowers, L. and Loga
Page 187 and 188: References 171 Turner, C. T., Davy,
Page 189 and 190: References 173 Vogt, R. G., Riddifo
Page 191 and 192: References 175 Widmayer, P., Heifet
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