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

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General Introduction 8 have been postulated based on experimental data and modelling of the perireceptor and receptor events involved in olfactory perception of moths (Kaissling, 2009). These include roles in the solubilisation and transport of ligands, protection of the ligands from enzymatic degradation by formation of OBP/ligand complex, interaction of the complex with specific ORs, or the OBP might be involved in the deactivation of the ligand. OBPs have been shown to be involved in the solubilisation of odorants in in vivo and in vitro assays. van den Berg and Ziegelberger (1991) perfused A. polyphemus sensilla trichodea with the pheromone (E,Z)-6,11-hexadecadienyl acetate dissolved in Ringer solution with or without PBP (ApolPBP) and showed that the pheromone concentration required to elicit a response from the ORN was decreased 100-fold in the presence of ApolPBP (van den Berg and Ziegelberger, 1991). As the pheromone solutions were applied to the ORN through a glass capillary, most of the pheromone that would have otherwise adhered to the capillary wall in the absence of the PBP would now be bound to the PBP hence remain in the soluble phase and be conferred to the ORN. This suggests a role for the A. polyphemus PBP in solubility and perhaps transport of the pheromone through the sensillum lymph to the ORN. In vitro binding assays have also been used to show a role for PBPs as solubilising agents for pheromones. Groβe-Wilde et al. (2006) generated stable cell lines expressing B. mori OR1 (BmOR1) in Human Embryonic Kidney (HEK293) cells. Functional assays where the ligand was solubilised in the organic solvent dimethyl sulfoxide (DMSO) showed that BmOR1 expressing cells elicited significant response to bombykol and bombykal. When the DMSO was replaced by BmPBP as the solubilising agent, BmOR1 expressing cells were able to elicit a response to bombykol with similar intensities (Große-Wilde et al., 2006). In another study the H. virescens pheromone receptor HvOR13, elicits a significant response to the main sex pheromone component Z11-hexadecenal when the pheromone is solubilised in DMSO, and this response sensitivity is increased by 10 4 -fold when the DMSO is replaced with HvPBP2, indicating roles of the PBP as a solubilising agent. The PBP might also be acting as a transporter, ferrying and increasing the local concentration of Z11-hexadecenal in close proximity to the PR (Große-Wilde et al., 2007).
General Introduction 9 OBPs have also been implicated in protecting the bound ligand from degradation. In A. polyphemus, a pheromone degrading enzyme (PDE) has been isolated and shown to effectively degrade the pheromone in vitro (Vogt et al., 1985). However, this activity is reduced in the presence of PBP, an indication that the PBP is protecting the pheromone from the PDE (Vogt and Riddiford, 1986). This is also supported by in vivo experiments in B. mori and A. polyphemus that show two different rates of pheromone degradation, with 17% of the pheromone degraded initially and the remaining 83% degraded at a slower rate. One explanation for this might be the formation of PBP/pheromone complex as the pheromone enters the sensillum lymph and its consequent protection from PDE (Kasang, 1971; Kasang and Kaissling, 1972; Kasang, 1973; Kasang et al., 1988; Kasang et al., 1989a; Kasang et al., 1989b). In vivo binding assays provide evidence that the pheromone/PBP complex interacts with the receptor molecules. Syed et al. (2006) expressed BmOR1 in the empty- neuron system, a mutant fly strain that is devoid of its native receptors in the ab3A neuron, and showed that receptor activity was achieved when the neuron was stimulated with bombykol, albeit with low sensitivity. This sensitivity was enhanced by the co-expression of the B. mori PBP, indicating that the PBP is involved in the pheromone/receptor interaction. Selective response of receptor neurons to pheromone components have been observed in the presence of different PBPs. A. polyphemus sensilla trichodea expressing the ORN tuned to the pheromone component (E,Z)-6,11- hexadecadienyl acetate elicited a response upon stimulation with the pheromone solubilised in DMSO, ApolPBP1 and ApolPBP3 (Pophof, 2004). However, no response was elicited when the pheromone was solubilised in ApolPBP2, giving further evidence of pheromone/PBP complex interaction with PR. Evidence for such pheromone/PBP complex interactions have also been shown in vivo and in vitro in B. mori. When solubilised in DMSO, both bombykol and bombykal bind BmOR1 but when DMSO is replaced with BmPBP, only the bombykol/BmPBP complex is able to activate BmOR1 (Große-Wilde et al., 2006). Interaction of the pheromone/PBP complex with receptor molecules has also been shown in flies. The Drosophila OBP LUSH has been suggested to interact directly with ORs (Kim et al., 1998). Flies mutant for LUSH were previously shown to have odour defects in that they were unable to avoid high concentrations of alcohol, as
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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 .....
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Page 15 and 16: List of Abbreviations gDNA Genomic
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The roles of Epiphyas postvittana G
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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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