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

More documents

Recommendations

Info

Concluding Discussion 128 discussed in section 4.4.1) indicating that phylogenetic relatedness does not necessitate functional conservation. The expression levels of 26 of the ORs were determined in male and female antennae by qRT-PCR as shown in Figure 4.5. EpOR30, 33 and 34 show high expression levels in male than female antennae (more than a ten-fold difference), making these good candidates for being PRs. These ORs, however, are not members of the PR clade hence it could be that the Tortricidae and Bombycidae ORs diverged early in lepidopteran evolution. If indeed these three ORs are the E. postvittana PRs, then it could be assumed that different ORs have been co- opted into a role in pheromone reception in E. postvittana. Twelve ORs were expressed in both male and female though the level of expression was higher in the male antennae, while 11 ORs were expressed at approximately similar levels in male and female antennae. No ORs with female antennae specific expression were detected. This could be because the ORs that were analysed for tissue expression were all from male antennae specific ESTs, while the ORs isolated from the genome scanning were not tested. Analysis of these ORs could possibly reveal the female- specific ORs. From a combination of the transcriptome, light genomics and 3‟RACE PCR, the full length coding region of EpOR34, one of the male antennal specific ORs was identified. Phylogenetic analysis showed that it shares 25 to 30% amino acid identity with BmOR18, 30, 33 and 34. Tissue expression analysis of the B. mori homologues of EpOR34 show that BmOR18 is expressed at similar levels in male and female antennae, BmOR30 is a female-specific receptor, and BmOR33 and 34 are expressed in both male and female antennae albeit at higher levels in male antennae (Wanner et al., 2007). However, due to the limited structural knowledge available for insect ORs and their low levels of homology, OR function based solely on homology cannot be predicted. 5.3 Current hypothesis and future directions The deorphaning of EpOR1 revealed it to be a receptor for plant volatiles. It will be interesting to see if and how genetic modification of EpOR1 affects recognition of its ligands by E. postvittana. EpOR1 and EpOR3 have been shown to share some ligands (Jordan et al., 2009), suggesting that in vitro one compound is recognised by more than one OR and vice versa, albeit with different levels of sensitivities. These ORs are
Concluding Discussion 129 redundant for some of their ligands, a phenomenon observed in Drosophila also (Hallem and Carlson, 2006). Perhaps the modification of EpOR1 will have no significant effect on odorant recognition by the moth. Such an observation would suggest a „backup‟ mechanism employed by moths; a survival advantage of having more than one protein recognising a particular compound. Or it could happen that the modification of one receptor affects the recognition of odorants by another receptor in vivo. Either way, such an experiment will contribute towards answering questions about olfactory mechanisms in insects. Gene silencing using RNA interference can be used for studying host localisation of E. postivittana, any impact will help in developing novel pest control strategies for this moth. EpOR1 could also be studied in the empty-neuron system and the results obtained from the in vitro Sf9 cell assays could be compared with the in vivo results to study if/how the ligand binding range of EpOR1 is affected. A number of studies have shown the involvement of moth PBPs in pheromone binding, however little data for insect GOBP role in odorant recognition exists. This study has demonstrated a role of EpGOBP2 in odorant solubilisation, and is the first documented study showing the involvement of a moth GOBP in odorant recognition by ORs in a heterologous expression system. The results are also indicative of a role of EpGOBP2 in odorant transport for example, a 100-fold decrease is observed in the concentration of geranyl acetate required to activate EpOR1 in the Sf9 cell assay (Figures 3.8B and C) in the presence of EpGOBP2. These results suggest that since OBPs have a role in OR-ligand binding, then other proteins identified in the olfactory system could play significant roles also. Odorant degrading enzymes could be introduced into the cell assay system in the presence and absence of EpGOBP2. If EpGOBP2 has a role in protecting ligands from degradation, then the introduction of ODE in the presence of EpGOBP2 will not have any effect on EpOR1 activation by its ligands. The interaction of EpGOBP2/ligand complex with OR can also be studied. Just like the reconstitution of the Sf9 cell assay system for EpOR1 with EpGOBP2, the effect of EpGOBP2 on ligand binding by EpOR3 could also be studied. This will reveal if and how the EpGOBP2/ligand complex interacts with the different ORs. The involvement of GOBPs in deactivation of ligands can also be investigated by studying effects of pH changes on GOBP structure. Future studies can look at decoding the roles played by PDEs and SNMPs in in vitro assay systems for ORs. Some cell
Page 1 and 2:
http://researchspace.auckland.ac.nz
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
Page 19 and 20:
General Introduction 3 moth in loca
Page 21 and 22:
General Introduction 5 Figure 1.1:
Page 23 and 24:
General Introduction 7 1.5 Componen
Page 25 and 26:
General Introduction 9 OBPs have al
Page 27 and 28:
General Introduction 11 A model has
Page 29 and 30:
Page 31 and 32:
General Introduction 15 Tanaka et a
Page 33 and 34:
General Introduction 17 Table 1.1:
Page 35 and 36:
Page 37 and 38:
General Introduction 21 et al., 200
Page 39 and 40:
General Introduction 23 the attract
Page 41 and 42:
General Introduction 25 leaves and
Page 43 and 44:
Page 45 and 46:
General Introduction 29 Expression
Page 47 and 48:
General Introduction 31 BmOr2 HvOr2
Page 49 and 50:
General Introduction 33 for lepidop
Page 51 and 52:
Functional characterisation of Epip
Page 53 and 54:
Page 55 and 56:
Page 57 and 58:
Page 59 and 60:
Page 61 and 62:
Page 63 and 64:
Page 65 and 66:
Page 67 and 68:
Page 69 and 70:
Page 71 and 72:
Page 73 and 74:
3.1 Introduction 3 The roles of Epi
Page 75 and 76:
The roles of Epiphyas postvittana G
Page 77 and 78:
Page 79 and 80:
Page 81 and 82:
Page 83 and 84:
Page 85 and 86:
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94: The roles of Epiphyas postvittana G
Page 95 and 96: 4.1 Introduction 4 Identification o
Page 97 and 98: Identification of putative odorant
Page 109 and 110: Microarray OR 454 Transcriptome OR
Page 141 and 142: 5.1 Introduction 5 Concluding Discu
Page 143: Concluding Discussion 127 VOBA sett
Page 147 and 148: Concluding Discussion 131 against t
Page 149 and 150: Concluding Discussion 133 sequencin
Page 151 and 152: Appendix B 135 Buffered TB (Sambroo
Page 153 and 154: Appendix C 137 C. Appendix C - Clus
Page 155 and 156: Appendix D 139 D. Appendix D - Solu
Page 157 and 158: Appendix E 141 I II
Page 159 and 160: Appendix E 143 V
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
show all

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

Create successful ePaper yourself

Delete template?

Save as template?