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General Introduction 14 sequence with candidate OR sequences from Drosophila as queries (Altschul et al., 1990; Krieger et al., 2002). The genomic sequences obtained from the BLAST search were used as probes for obtaining the exon regions of the sequences from a H. virescens cDNA library. This process identified nine OR genes in H. virescens and further screening of the genomic sequences as well as transcriptome sequencing have led to the identification of a total of 18 ORs in H. virescens so far (Krieger et al., 2004). Five of these ORs (HvOR11, HvOR13–HvOR16) have been identified as potential PRs of H. virescens based on the high levels of expression of these genes in male than female antennae (Krieger et al., 2004; Vásquez et al., 2010). In situ hybridisations of HvOR11, HvOR13, HvOR14 and HvOR16 showed their expression was confined to antennal cells below the sensilla trichodea (Große-Wilde et al., 2007; Baker, 2009; Krieger et al., 2009) and functional characterisation in Xenopus laevis oocytes has confirmed HvOR13, HvOR14 and HvOR16 to bind (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate and (Z)-11-hexadecen-1-ol respectively (Wang et al., 2010). (Z)-9-tetradecenal forms 5% of the sex pheromone component of H. virescens and HvOR6, which is expressed in both male and female antennae, and was identified as the receptor for this pheromone component by expression and characterisation in X. laevis oocytes (Wang et al., 2010). The H. virescens ORs share very little homology to other known insect ORs, except for HvOR2, which is highly conserved across insects and has a role as a co-receptor in olfactory signalling (refer to section 1.5.2.2 for details of this receptor type). Digoxigenin (DIG) labelled probes designed to HvOR2 were used to isolate the first ORs from B. mori and Antheraea pernyi antennal cDNA libraries (Krieger et al., 2003). Screening of a B. mori male antennal cDNA library as well as the B. mori genome sequences in GenBank with other H. virescens OR sequences led to the identification of a total of six ORs (Sakurai et al., 2004; Krieger et al., 2005). In situ hybridisations has revealed BmOR1 and BmOR3 to be expressed in the sensilla trichodea, while BmOR2 has dispersed expression not confined to the sensilla trichodea, and BmOR4 and BmOR5 are expressed in fewer cells. BmOR6 did not label any cells. Heterologous expression of BmOR1 and BmOR3 in X. laevis oocytes have identified their ligands as bombykol and bombykal, respectively (Nakagawa et al., 2005). The availability of the B. mori genome sequence has facilitated the identification of a total of 68 putative ORs (Sakurai et al., 2004; Wanner et al., 2007;
General Introduction 15 Tanaka et al., 2009), and tissue expression analysis has identified four ORs (BmOR19, BmOR30, BmOR45 and BmOR47) with higher expression levels in female than male antennae (Anderson et al., 2009). In situ hybridisation has shown BmOR19 to be co-localised with either BmOR45 or BmOR47. Heterologous expression of three of these ORs in Sf9 insect cells has identified BmOR19 to be tuned to linalool, and BmOR45 and BmOR47 to be tuned to benzoic acid, 2- phenylethanol and benzaldehyde (Anderson et al., 2009). Mitsuno et al. (2008) identified ten ORs from three different moth species; Plutella xylostella (PxOR1, PxOR2, PxOR3 and PxOR4), Mythimna separate (MsOR1, MsOR2 and MsOR3), and Diaphania indica (DiOR1, DiOR2 and DiOR3), through degenerate PCR of male antennal cDNA of the moths with primers designed from the conserved regions of BmOR1 and other male-specific ORs. Three of the ORs were homologues of the highly conserved BmOR2. Five of the ORs (PxOR1, PxOR4, DiOR1, MsOR1 and MsOR3) were expressed exclusively in male antennae, while two (PxOR3, DiOR3) were expressed in both male and female antennae, although higher levels were observed in male antennae. PxOR1, MsOR1 and DiOR1 were exclusively expressed in male antennae and shown to bind sex pheromone component(s) of the respective moths (P. xylostella binds (Z)-11-hexadecenal; M. separate binds (Z)-11-hexadecenyl acetate and D. indica binds (E)-11-Hexadecenal) (Mitsuno et al., 2008). M. sexta ORs were identified by differential screening of a male antennal cDNA library (OR1), degenerate PCR (OR2, which is the homologue of BmOR2) and from a subtracted male antennal transcriptome (OR3) (Patch et al., 2009). Tissue expression analysis revealed MsexOR1 to be expressed in male antennae and MsexOR3 to be expressed highly in female antennae. Three further ORs, named MsexOR4, MsexOR5 and MsexOR6 have been identified in M. sexta recently from a subtractive male antennal cDNA library (Große-Wilde et al., 2010). MsexOR4 has been shown to be expressed in male antennae while MsexOR5 and MsexOR6 have been detected in female antennae. Functional analysis of MsexOR1 with pheromone components did not reveal any ligands for this receptor as yet and MsexOR4 has been postulated to bind one of the sex pheromone components of M. sexta however, no functional data for odorant binding of these ORs are available as yet.
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Page 3 and 4: Abstract Abstract Olfaction or the
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
Page 13 and 14: List of Tables List of Tables Table
Page 15 and 16: List of Abbreviations gDNA Genomic
Page 17 and 18: 1.1 General overview 1 General Intr
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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 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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