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

More documents

Recommendations

Info

Functional characterisation of Epiphyas postvittana odorant receptor 1 52 to the low transfection rate of the ORs in Sf9 cells, a major drawback of this assay system (Kiely, 2008). The expression of the OR by the Sf9 cells is random, with only about 5–10% of the cells being transfected and the percentage of these transfected cells in the chosen field of view can vary anywhere between 0 to 100%. Therefore, a large number of assays have to be conducted to obtain meaningful data as well as assign a test odorant as a responder or a non-responder. In Figure 2.3, not all the cells show the same fluorescent levels upon the addition of the ionophore, ionomycin. This is due to the differential uptake of Fluo-4 by the Sf9 cells, therefore each cell in the field of view is selected manually and analysed using a semi-automated excel algorithm (Kiely, 2008). E. postvittana OR1 is activated by ten of the 33 compounds it was tested with. These compounds were chosen either due to their ability to elicit an EAG response in whole antennae of E. postvittana (Suckling et al., 1996), or their occurrence as plant semiochemicals. The major sex pheromone component of E. postvittana, E-11- tetradecenyl acetate, was also tested but no response was obtained, suggesting a role of this receptor as a general OR and not a PR. This conclusion is consistent with the qRT-PCR data obtained in Jordan et al. (2009) which showed no sex bias of this receptor in male and female antennal tissue. To date, all moth PRs that have been characterised show male biased expression (Krieger et al., 2004; Sakurai et al., 2004; Mitsuno et al., 2008; Forstner et al., 2009; Patch et al., 2009). These two results are, however, in contrast with phylogenetic data where EpOR1 belongs to a cluster of mostly sexually dimorphic ORs from five other moths, most of which are male biased PRs (Figure 1.8) (Krieger et al., 2004; Sakurai et al., 2004; Mitsuno et al., 2008; Jordan et al., 2009). The amino acid identity within this clade is only about 13%, with EpOR1 having an average of 35% sequence identity with individual OR sequences. EpOR1 has the highest amino acid sequence identity (40%) with D. indica OR1 (DiOR1), which is a member of Lepidoptera:Crambidae. Two-electrode voltage clamp recordings of X. laevis oocytes transiently expressing DiOR1 show this receptor to be most sensitive to the major sex pheromone component for this moth, E11-16: aldehyde (Mitsuno et al., 2008). Even though EpOR1 and DiOR1 are closely related phylogenetically, the ligands they bind are very different implying that phylogenetic relatedness does not indicate functional conservation in evolutionary diverse moths.
Functional characterisation of Epiphyas postvittana odorant receptor 1 53 EpOR1 is highly sensitive, recognising odorants at very low concentrations. For example, EpOR1 can recognise methyl salicylate at a concentration as low as 10 -15 M, with an EC50 of 1.8 x 10 -12 M when transiently expressed in Sf9 cells (Figure 2.4A). This sensitivity of insect ORs has been shown in other studies of ORs expressed in Sf9 cell lines. Jordan et al. (2009) has shown that another OR from E. postvittana, EpOR3 binds citral at concentrations as low as 10 -15 M when expressed in Sf9 cells. Two other studies showed high sensitivity of ORs to their respective ligands when expressed in Sf9 cells. Kiely et al. (2007) showed D. melanogaster OR22a can respond to ethyl butyrate at the low concentration of 10 -13 M with an EC50 of 1.58 x 10 -11 M, while Anderson et al. (2009) showed the B. mori female specific OR, BmOR47 can respond to benzoic acid at 10 -14 M with an EC50 of 1.42 x 10 -11 M. This sensitivity of insect ORs is maintained even when the transiently expressing cell line is of non-insect origin. B. mori OR1 expressed in HEK293 cells bind bombykol to concentrations of 10 -12 M with EC50 of 10 -10 M (Große-Wilde et al., 2006) while the pheromone receptor from H. virescens HvOR13 bind to the major sex pheromone component of this moth in the presence of pheromone binding protein at a concentration of 10 -14 M with an EC50 of 2 x 10 -13 M (Große-Wilde et al., 2007). However, while highly sensitive ORs with different kinetics for various odorants are seen in the Sf9 cell assay systems in both moths and flies, the response profile might be very different in a biological context. Inferences on how the concentration of certain odorants affect the behaviour of the moth in the environment cannot be fully justified based on this in vitro data and behavioural studies need to be conducted to reconcile these differences. The dose response of EpOR1 to different odorants can either be „gently sloping‟ where EpOR1 response increases gradually with the concentration of the compound, (for example, the dose response curves of methyl salicylate, citral, geranyl acetate and geranial show this effect; these gradual change can be attributed as a concentration effect; as the concentration of the ligand increases, the fluorescence of the cells increases similarly); or „abrupt‟ where the odorant acts like a „switch‟, turning the OR „on/off‟ at a particular concentration (for example, there is a sharp increase in the binding of geraniol to EpOR1 from 10 -11 M to 10 -10 M in Figure 2.4D. It appears that a concentration of 10 -10 M acts as a switch that turns on the receptor). This difference in
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 31 and 32: General Introduction 15 Tanaka et a
Page 33 and 34: General Introduction 17 Table 1.1:
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 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 67: Functional characterisation of Epip
Page 73 and 74: 3.1 Introduction 3 The roles of Epi
Page 75 and 76: 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 119 and 120:
Identification of putative odorant
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
Page 129 and 130:
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Page 139 and 140:
Page 141 and 142:
5.1 Introduction 5 Concluding Discu
Page 143 and 144:
Concluding Discussion 127 VOBA sett
Page 145 and 146:
Concluding Discussion 129 redundant
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 ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?