Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...

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General Introduction 28 Table 1.2: Plant volatiles detected by E. postvittana in electroantennogram experiments from (Suckling et al., 1996). Plant Volatiles Citral S-(-)-β-Pinene Z-3-Hexanol E-2-Hexenal Nonanol Octanol Phytol Hexanal α-Farnesene E-Caryophyllene Nonanal Heptanol Eugenol Thymol (+)-Limonene Heptanal Linalool (-)-α-Copaene Z-3-Hexyl acetate Acetophenone n-Decyl aldehyde Carvacrol Tetradecanol (-)-Limonene Geraniol α-Terpineol Farnesol Benzaldehyde 1-Indanone Nerolidol A molecular based approach has been pursued into the odorant coding mechanism employed by this moth. Newcomb et al. (2002) isolated four OBPs from male and female antennae of E. postvittana. Native polyacrylamide gel electrophoresis (PAGE) separated four OBPs and the full sequence was then obtained by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using primers based on N- terminal amino acid sequencing (Newcomb et al., 2002). Two of the OBPs had sequence similarity to other lepidopteran PBPs, while two were GOBPs. The two E. postvittana PBPs differed by six amino acid substitutions and were found on the same gene, hence they were two alleles of the same gene, however due to differential mobility‟s on native protein gel, these were named PBP fast and PBP slow. Both these PBPs bound to radio-labelled major pheromone component E11-14:OAc in gel based assay and hence confirmed them as PBPs (Newcomb et al., 2002). Upon further sequence analysis, one of the GOBPs has been classified as a second PBP, PBP2.
General Introduction 29 Expression and two-dimensional gel electrophoresis (2D-GE) separation of antennal proteins revealed other soluble proteins in the antennae of both the male and female moths. 26 proteins were revealed, 17 of these proteins were expressed more in male antennae than in female (Jordon, 2006). The creation and screening of a male E. postvittana antennal cDNA EST library revealed several chemosensory proteins and takeout proteins (Jordan et al., 2008; Hamiaux et al., 2009). Three putative olfactory receptors were identified from this EST database through phylogenetic analysis (Jordan et al., 2009). Full length sequences of these receptors was obtained via 5‟RACE and the amino acid sequence identity of these receptors with other known insect ORs was investigated. The receptor named EpOR1 has seven predicted transmenbrane domains, a coding region of 1245 nucleotides and a predicted protein of 415 amino acids. Homology search of this OR with other insects showed that it had 36% sequence identity to B. mori OR1, a pheromone receptor (Figure 1.8). However, tissue expression analysis showed similar levels of expression in male and female antennae, indicating that EpOR1 might not be a pheromone receptor; and functional analysis using calcium imaging of this receptor in Spodoptera frugiperda 9 (Sf9) cells did not show any response to the E. postvittana sex pheromone. The N– and C– termini of EpOR1 were c-Myc-epitope-tagged to determine the membrane topology (Jordan et al., 2009). The N-terminus was only accessible in the presence of saponin while the C-terminus was accessible regardless of saponin presence, indicating an intracellular N-terminus and an extracellular C- terminus, consistent with the membrane orientation observed for Drosophila Or83b and Or22a. The second putative receptor, EpOR2 has a coding region of 1422 nucleotides and the predicted protein is 474 amino acids. This putative receptor has a long 3‟ untranslated region (UTR), as compared with EpOR1. Homology search revealed high sequence identity to the Drosophila co-receptor Or83b and 84% identity with BmOR2. Tissue expression analysis indicated EpOR2 to be expressed in both male and female antennae, with a higher level of expression in male antennae. Consistent with being the co-receptor in E. postvittana, EpOR2 is expressed about 13–57 times more highly than EpOR1 and the third receptor, EpOR3. The third putative receptor EpOR3 has a coding region of 1230 nucleotides and a predicted protein of 410 amino acids. This receptor has 65% sequence identity with BmOR49J from B. mori (Anderson et al., 2009; Jordan et al., 2009) [in Anderson et al. (2009),
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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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Page 39 and 40: General Introduction 23 the attract
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Page 47 and 48: General Introduction 31 BmOr2 HvOr2
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Page 51 and 52: Functional characterisation of Epip
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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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