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Identification of putative odorant receptors from Epiphyas postvittana 122 Another clade of interest is the female-specific odorant receptor clade. Members of this lineage have been identified in B. mori and M. sexta, and have been shown to have female-specific tissue expression (Wanner et al., 2007; Anderson et al., 2009; Große-Wilde et al., 2010). MsOR5 forms a lineage with BmOR19 while BmOR45, 46, 47, 48 and 50 cluster together phylogenetically. EpOR46 shares 17% and EpOR50 shares 20% amino acid identity with BmOR47 and BmOR46 respectively. The low level of homology between the E. postvittana homologues of the B. mori female biased clade suggest that either the E. postvittana OR repertoire is incomplete and the female biased ORs of this moth have not been identified yet, or the E. postvittana female biased ORs are not phylogenetically related to the B. mori homologues and only tissue expression analysis of all of the E. postvittana ORs will identify the female biased ORs. The tissue expression of 26 of the E. postvittana ORs in male antennae, female antennae and body tissues revealed differing levels of expression both between the various ORs in the same tissue as well as of the same OR in different tissues. The expression levels of these putative ORs were assessed for male-biased expression as it was assumed that a sex pheromone receptor would be more highly expressed in the male compared with the female antennae as found in various lepidopterans (Krieger et al., 2004; Krieger et al., 2005; Mitsuno et al., 2008; Große-Wilde et al., 2010). Three of the 26 candidate ORs identified from the deep transcriptomics displayed male- biased expression. EpOR34 had 1500 times higher expression in male antennae than female antennae, and EpOR30 and EpOR33 had 600 times and 900 times higher expression, respectively (Figure 4.5). Sex biased expression of ORs is a good indicator of their involvement in sex specific odour recognition as supported by evidence from B. mori ORs (Sakurai et al., 2004). BmOR1 and BmOR3 have male biased expression (with upto10,000 times higher expression in male antennae than in female) and have been shown in a number of in vivo studies as well as in heterologous assays to be involved in sex-specific pheromone recognition (Sakurai et al., 2004; Nakagawa et al., 2005; Große-Wilde et al., 2006; Syed et al., 2006; Wanner et al., 2007). It is tempting to correlate the high expression levels of EpOR30, 33 and 34 in male antennae with roles in recognising the three components of the E. postvittana sex pheromone, however, further functional analysis of these ORs will be crucial in
Identification of putative odorant receptors from Epiphyas postvittana 123 identifying the roles of these three male-biased ORs in E. postvittana olfaction. ORs with high expression levels in female antennae have been shown to be involved in recognition of odorants that elicit female-specific behaviours in moths. For example, in B. mori, BmOR19 has 830 times higher expression in female antennae and functional analysis revealed it to be the receptor for linalool, a major constituent of mulberry leaves which serve as oviposition sites for B. mori (Anderson et al., 2009). No such female-specific ORs have been identified from tissue expression analysis of E. postvittana ORs as yet. This could be due to the fact that the tissue expression of the putative E. postvittana ORs have only been carried out on the ORs identified from the deep transcriptomics of male antennal cDNA. This sample is rich in RNA expressed highly in male antennae compared with female antennae hence the identification of any female-specific ORs in this sample will be minimal. The female- specific ORs would be more likely identified from low coverage whole genome sequences. Tissue expression analysis of the 23 putative ORs identified exclusively by the low coverage whole genome sequencing should reveal the female-specific ORs of E. postvittana, if any. Twelve ORs showed expression in both male and female antennae, although the expression level in the male antennae was higher than in female. These male biased ORs might have a role in detecting odorants that control eminent behaviours in male moths. Female moths have been shown to be able to detect female released sex pheromones hence the expression of male biased ORs in females could be involved in the detection of sex pheromones (Widmayer et al., 2009). Eleven of the 26 putative E. postvittana ORs have similar levels of expression in both male and female antennae, indicating their role in detecting odorants such as plant volatiles to locate host plants. Eight ORs (EpOR7, 13, 24, 27, 33, 49, 50 and 52) were also detected in the body. Studies in H. virescens has revealed the expression of ORs (HvOR2, 6 and 13) in the abdomen of this moth. One hypothesis is that the female moth may be detecting its own sex pheromone in a negative feedback mechanism to control its release into the environment. ORs such as EpOR27, 39 and 48–52 have upto 100-fold lower expression levels as compared with EpOR1, 2, 3, 13, 33, 34, 36 and 42 in the moth tissues, probably due to their expression restricted to a few sensilla, or they are more highly expressed at other developmental life stages (Tanaka et al., 2009). This varied range of expression levels of different ORs are also seen in B. mori (Wanner et al., 2007).
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
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Contents 4.2.5 Degenerate PCR .....
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List of Figures List of Figures Fig
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List of Tables List of Tables Table
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List of Abbreviations gDNA Genomic
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1.1 General overview 1 General Intr
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General Introduction 3 moth in loca
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General Introduction 5 Figure 1.1:
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General Introduction 7 1.5 Componen
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General Introduction 9 OBPs have al
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General Introduction 11 A model has
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General Introduction 15 Tanaka et a
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General Introduction 17 Table 1.1:
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General Introduction 21 et al., 200
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General Introduction 23 the attract
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General Introduction 25 leaves and
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General Introduction 29 Expression
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General Introduction 31 BmOr2 HvOr2
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General Introduction 33 for lepidop
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Functional characterisation of Epip
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3.1 Introduction 3 The roles of Epi
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The roles of Epiphyas postvittana G
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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,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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