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The School of Biological Sciences
The University of Auckland
New Zealand
Identification and
Analysis of Olfactory
Receptors from the Light
Brown Apple Moth,
(Epiphyas postvittana)
Doreen Shabana Begum
March 2011
Supervisors: Richard D. Newcomb
Andrew V. Kralicek
David L. Christie
A thesis submitted in fulfilment of the requirements for the degree of Doctor of
Philosophy in Biological Sciences, The University of Auckland, 2011.

Abstract
Abstract
Olfaction or the sense of smell is one of the major modes of communication in moths,
playing an integral part in the moths‟ ability to locate mates for reproduction, location
of host plants and oviposition sites. Some of the key players of the moths‟ olfactory
system include general odorant binding proteins and olfactory receptors (OR). The
aim of this study was to investigate the molecular mechanisms of olfaction in the light
brown apple moth, Epiphyas postvittana. These data will contribute to the
development of new pest control strategies for the moth and the development of
olfactory biosensors. E. postvittana OR1 (EpOR1) is closely related to the pheromone
receptors (PR) of other lepidopterans, but is expressed at similar levels in male and
female antennae. Functional analysis in Sf9 cells demonstrated that it binds important
plant semiochemicals but not pheromone. EpOR1 is sensitive to methyl salicylate,
recognising this odorant to a low concentration of 10 -15 M. EpOR1 also binds a range
of terpenes which showed Hill slopes ranging from 0.33–3.2.
E. postvittana GOBP2 (EpGOBP2) was expressed in bacteria and purified to
homogeneity. Binding analysis revealed it was able to bind octanol, α-farnesene,
methyl salicylate, nerol, eucalyptol, geranyl acetate and pentyl acetate, but not citral,
geranial and geraniol. EpGOBP2 was able to replace DMSO as a solubilising agent
for methyl salicylate and geranyl acetate in a functional assay of EpOR1 in Sf9 cells.
The sensitivity of EpOR1 for these two ligands in vitro was enhanced in the presence
of EpGOBP2 [EC50 of EpOR1 with methyl salicylate in DMSO is (1.8 ± 0.9) x 10 -
12 M and EC50 of EpOR1 with methyl salicylate in the presence on EpGOBP2 is (1.19
± 0.82) x 10 -13 M], suggesting that EpGOBP2 might not only be acting as a
solubilising agent but also as an activated ligand.
Forty-nine new ORs were identified by deep transcriptomic sequencing and light
coverage genome scanning of E. postvittana bringing the total number of ORs
identified to date for this species to 52. Phylogenetic analysis revealed that associated
with lineage, 24 ORs had one-to-one orthologs with ORs from the silkworm, Bombyx
mori, while 28 E. postvittana ORs were expansions compared with B. mori. EpOR1
i

Abstract
and EpOR6 are the most closely related E. postvittana ORs to other moth pheromone
receptors. However, EpOR1 binds plant semiochemicals and not pheromone
components and EpOR6 remains to be functionally annotated. Tissue expression
analysis of 26 of the ORs revealed that three (EpOR30, 33 and 34) are more than 600
times more highly expressed in male than female antennae, making them good
candidates for being the pheromone receptors of E. postvittana. Functional analysis of
these ORs will reveal their role(s) in E. postvittana olfaction.
ii

Acknowledgements
Acknowledgements
I would like to extend my sincere thanks for the guidance and support I received from
my supervisors: Richard Newcomb, Andrew Kralicek and David Christie. This PhD
would not have been possible without your encouragement and open door policy for
us students. Thank you for always being available for discussions from the
commencement through to the completion of my research.
No work is complete without the efforts of all the members of a team. My heartfelt
thanks to all the members of the Molecular Sensing Team. Thank you Melissa for
doing the groundwork on EpOR1; to Cyril for your expertise in recombinant protein
expression and purification; to Colm and Aidan for sharing your expertise of Sf9 cell
culture; to Nadeesha for your guidance in the lab and for your friendship and to
Edwige for your valuable time and guidance in dealing with the ghosts of qRT-PCR,
and your friendly support and encouragement in the highs and lows of my life. A big
thank you to Pablo, Andy, Sylvia, Jeremy, Leah and Caroline for your company in the
lab and during the many lunch breaks. The time spent as part of the Molecular
Sensing team would not have been enjoyable without you all!
A very big thank you to Anne Barrington of the insect rearing facility at Plant and
Food Research Ltd, for rearing the thousands of sacrificial moths without whom a
major part of this thesis would have been incomplete. Thank you to the members of
the microarray facility at Plant and Food Research, especially Luke Luo for setting up
the hybridisations and Bart Jenssen for your expertise on data analysis. I would also
like to show my gratitude to the Bioinformatics department at Plant and Food
Research, especially Ross Crowhurst for doing all the raw data analysis of the tons of
sequencing with a smile! Thank you a lot Ross!
I would also like to thank the Allan Wilson Centre Genome Service, especially
Lorraine Berry for your advice on sample preparations and genomic sequencing; and
to the team at the University of Otago high-throughput DNA sequencing unit.
iii

Acknowledgements
I owe my deepest gratitude to my family, without whose support and encouragement
this achievement would never have been possible. Thank you mum and dad for
always encouraging me in my studies; to my siblings Shaireen, Imtiyaz, Imraan and
Rizwaan for sharing in my life. I would also like to thank my parents-in-law and
Arisha for your support, and to Sohail and Shayaan for being the brightest stars in my
life. A very big thank you to my husband Aznain for your understanding and
sacrifices during the course of my project, especially for your patience and words of
guidance during the times when writing seemed like an unattainable feat. It just would
not have been possible without all your love, support and encouragement.
Finally, I would like to thank School of Biological Sciences and The University of
Auckland for the travel grants for supporting my attendance to the Queenstown
Molecular Biology Meeting (2006), to the International Symposium of Taste and
Olfaction (2008) and to the Genetics Society of Australasia (2010). This research has
been made possible through grants from Foundation of Research, Science and
Technology and the Ministry of Research, Science and Technology of New Zealand.
iv

Contents
v
Contents
Abstract……………………………………………………………………...…...…...i
Acknowledgements…………………………………………………………….……iii
List of Figures……………………………………………………………………..…ix
List of Tables…………………………………………………………………...……xi
List of Abbreviations…………………………………………………………...…..xii
1 General Introduction ........................................................................................... 1
1.1 General overview ............................................................................................ 1
1.2 Olfaction in insects .......................................................................................... 2
1.3 Olfaction in moths ........................................................................................... 2
1.3.1 Sex pheromones ....................................................................................... 2
1.3.2 Other odorants detected by moths............................................................ 3
1.4 The olfactory system ....................................................................................... 4
1.5 Components of the olfactory system ............................................................... 7
1.5.1 Proteins found in the sensillum lymph..................................................... 7
1.5.2 Membrane proteins ................................................................................ 12
1.5.2.1 Odorant receptor identification in moths ........................................... 13
1.5.2.2 Insect odorant receptor activation ...................................................... 18
1.6 Insect pest control strategies ......................................................................... 21
1.7 Light brown apple moth ................................................................................ 24
1.8 Aims .............................................................................................................. 32
2 Functional Characterisation of Epiphyas postvittana Odorant Receptor 1 .. 34
2.1 Introduction ................................................................................................... 34
2.1.1 Aims ....................................................................................................... 39
2.2 Materials and methods .................................................................................. 39
2.2.1 Materials ................................................................................................ 39
2.2.2 Insect cell culture ................................................................................... 40
2.2.3 Transfection of cells ............................................................................... 40
2.2.4 Detection of mRNA encoding pIB-EpOR1 by RT-PCR ....................... 41
2.2.5 Membrane Fraction Isolation and Western Blot .................................... 41
2.2.6 EpOR1 functional assay ......................................................................... 43
2.2.7 Data analysis .......................................................................................... 44

Contents
2.3 Results ........................................................................................................... 45
2.3.1 EpOR1 functional characterisation in Sf9 cells ..................................... 46
2.4 Discussion ..................................................................................................... 51
3 The roles of Epiphyas postvittana GOBP2 in odour detection by EpOR1 .... 57
3.1 Introduction ................................................................................................... 57
3.1.1 Aim ........................................................................................................ 60
3.2 Materials and methods .................................................................................. 61
3.2.1 Recombinant Expression of EpGOBP2 ................................................. 61
3.2.1.1 Purification of recombinant EpGOBP2.............................................. 62
3.2.1.2 Delipidation of recombinant EpGOBP2 ............................................ 62
3.2.2 Volatile odorant binding assay............................................................... 63
3.2.3 Reconstituted cell assay ......................................................................... 65
3.3 Results ........................................................................................................... 65
3.3.1 Purification of recombinant GOBP2 ...................................................... 65
3.3.2 Volatile Odorant Binding Assay ............................................................ 68
3.3.3 Reconstituted EpOR1 receptor activation assays .................................. 70
3.4 Discussion ..................................................................................................... 74
4 Identification of putative odorant receptors from Epiphyas postvittana ....... 79
4.1 Introduction ................................................................................................... 79
4.1.1 Transcriptome sequencing – EST approach .......................................... 79
4.1.2 Genome sequencing ............................................................................... 80
4.1.3 Recent progress in sequencing field ...................................................... 81
4.1.3.1 454 Sequencing .................................................................................. 82
4.1.3.2 Illumina Genome Analyzer ................................................................ 82
4.1.3.3 Bioinformatics .................................................................................... 82
4.1.4 Odorant Receptor Identification in Moths ............................................. 83
4.1.5 Aims ....................................................................................................... 85
4.2 Materials and Methods .................................................................................. 86
4.2.1 Materials ................................................................................................ 86
4.2.2 Total RNA Extraction ............................................................................ 86
4.2.3 mRNA Isolation ..................................................................................... 86
4.2.4 cDNA Synthesis ..................................................................................... 86
vi

Contents
4.2.5 Degenerate PCR ..................................................................................... 86
4.2.6 Cloning and Sequencing ........................................................................ 87
4.2.7 Microarray Screening............................................................................. 88
4.2.8 Microarray Data Analysis ...................................................................... 89
4.2.9 Transcriptome Sequencing..................................................................... 89
4.2.10 Genome Sequencing .............................................................................. 90
4.2.11 Nuclear DNA isolation and mitochondrial DNA contamination test .... 90
4.2.11.1 Isolation of insect nuclei ................................................................. 90
4.2.11.2 Genomic DNA extraction ............................................................... 91
4.2.11.3 Mitochondrial DNA contamination test ......................................... 91
4.2.12 qRT-PCR primer design ........................................................................ 92
4.2.13 qRT-PCR primer test ............................................................................. 94
4.2.14 Quantitative Real-Time PCR ................................................................. 94
4.2.15 Rapid Amplification of cDNA Ends (RACE) ....................................... 96
4.2.16 Bioinformatics........................................................................................ 98
4.3 Results ........................................................................................................... 99
4.3.1 Microarray Analysis............................................................................... 99
4.3.2 Deep Transcriptomics .......................................................................... 100
4.3.2.1 454 Sequencing Statistics ................................................................. 101
4.3.3 Mitochondrial DNA contamination ..................................................... 102
4.3.4 Illumina Sequencing ............................................................................ 103
4.3.5 Data Mining ......................................................................................... 103
4.3.6 Quantitative Real-Time PCR ............................................................... 110
4.3.7 EpOR34 – a putative PR ...................................................................... 114
4.4 Discussion ................................................................................................... 119
5 Concluding Discussion ..................................................................................... 125
5.1 Introduction ................................................................................................. 125
5.2 Summary of results and discussion ............................................................. 126
5.2.1 EpOR1 characterisation ....................................................................... 126
5.2.2 EpGOBP2 reconstitution of the Sf9 cell assay .................................... 126
5.2.3 Identification of putative ORs from E. postvittana .............................. 127
5.3 Current hypothesis and future directions .................................................... 128
A. Appendix A – Dose response profile of pIB-V5 His..................................134
vii

Contents
B. Appendix B – EpGOBP2 expression buffer recipe and protein details ...... 135
C. Appendix C – ClustalX multiple sequence alignment of moth PR clade .... 137
D. Appendix D – Solutions for nuclear DNA isolation ...................................... 139
E. Appendix E – Multiple sequence alignment of E. postvittana ORs.............. 140
References ................................................................................................................. 146
viii

List of Figures
List of Figures
Figure 1.1: Structure of a single insect olfactory sensillum ......................................... 5
Figure 1.2: Odorant receptor activation ...................................................................... 10
Figure 1.3: Model of insect pheromone detection ...................................................... 13
Figure 1.4: Topologies of insect and mammalian odorant receptors .......................... 19
Figure 1.5: The ion channel–GPCR model of odorant signalling .............................. 20
Figure 1.6: The sex pheromone blend of E. postvittana. ............................................ 26
Figure 1.7: Scanning electronmicrograph of adult male (left) and female (right)
antennaes of E.postvittana. .......................................................................................... 27
Figure 1.8: Phylogenetic tree constructed from lepidopteran odorant receptors with
the three E. postvittana ORs ........................................................................................ 31
Figure 2.1: Reverse transcription PCR analysis ......................................................... 45
Figure 2.2: Western blot of myc-tagged EpOR1 ........................................................ 46
Figure 2.3: The change in response of a single Sf9 cell transfected with pIB-EpOR1
over the timecourse of a calcium assay experiment. .................................................... 47
Figure 2.4: Dose response curves ............................................................................... 50
Figure 3.1: Schematic representation of the VOBA setting. ...................................... 64
Figure 3.2: Purification of His6-EpGOBP2 by HiTrap chelating HP column. ....... 66
Figure 3.3: Purification of His6-EpGOBP2 from the pooled HiTrap fractions by Q-
sepharose anion exchange chromatography................................................................. 67
Figure 3.4: Purification of AcTEV cleaved EpGOBP2 .............................................. 68
Figure 3.5: Overnight VOBA of EpGOBP2 ............................................................... 69
Figure 3.6: Dissociation constants ( ) for ten odorants against EpGOBP2 ............. 69
Figure 3.7: Dose response of EpOR1 expressing Sf9 cells to EpGOBP2 .................. 71
Figure 3.8: Dose response curves of EpOR1 to geranyl acetate ................................. 72
Figure 3.9: Dose response curves of EpOR1 to methyl salicylate.............................. 73
Figure 4.1: Quantitative RT-PCR of four candidates PRs .......................................... 99
Figure 4.2: E. postvittana male antennal SMART-amplified cDNA ....................... 101
Figure 4.3: Semi-quantitative PCR on nuclear (Takeout 3) and mitochondrial
(cytochrome oxidase I) genes .................................................................................... 103
Figure 4.4: Multiple sequence alignment in ClustalX of E. postvittana ORs.......... 110
Figure 4.5: Expression levels of the putative E. postvittana ORs ............................ 111
ix

List of Figures
Figure 4.6: Phylogram resulting from multiple sequence alignment........................ 113
Figure 4.7: EpOR34 cDNA nucleotide sequence ..................................................... 115
Figure 4.8: Schematic representation of the consensus of the predicted TM regions
from seven TM prediction programs ......................................................................... 116
Figure 4.9: Transmembrane domain prediction of EpOR34 with TMHMM 2.0
algorithms .................................................................................................................. 117
Figure 4.10: Transmembrane domain prediction of EpOR34 with TMMOD
algorithms. ................................................................................................................. 117
Figure 4.11: Theoretical membrane topology of EpOR34 ....................................... 118
Figure A.1: Dose response of the empty pIB-V5 His...............................................134
Figure B.1: Chromatogram showing the elution profile of His6-EpGOBP2 from
HiTrap chelating HP column. ................................................................................ 136
Figure B.2: Elution profile of His6-EpGOBP2 on Q-sepharose HP column. ........... 136
Figure C.1: ClustalX multiple sequence alignment of the moth PR clade ............... 138
Figure E.1: Multiple sequence alignment in ClustalX of E. postvittana ORs with
those of five other species .......................................................................................... 145
x

List of Tables
List of Tables
Table 1.1: Summary of the moth pheromone receptors .............................................. 17
Table 1.2: Plant volatiles detected by E. postvittana in electroantennogram
experiments from (Suckling et al., 1996). ................................................................... 28
Table 2.1: Change in fluorescence at 10 -5 M of the compounds tested with EpOR1 in
the Sf9 cell assay. ......................................................................................................... 48
Table 2.2: EC50 (± standard error) and Hill slope estimates of the dose response
curves of five best ligands for EpOR1 ......................................................................... 51
Table 3.1: A comparison of the EC50 values ............................................................... 74
Table 4.1: Outer and nested degenerate primers used in degenerate PCR of male E.
postvittana antennal cDNA. ......................................................................................... 87
Table 4.2: Primer pairs for qRT-PCR amplification ................................................... 93
Table 4.3: Gene specific primers used for amplifying 3‟RACE products. ................. 96
Table 4.4: Primers used for amplifying 5‟ RACE products ........................................ 98
Table 4.5: Analysis of the 454 transcriptome sequence data .................................... 102
Table 4.6: E. postvittana OR repertoire to date ........................................................ 104
Table D.1: Reagents for making 1 L of Nuclei Extraction Buffer, pH 6. ................. 139
xi

List of Abbreviations
ΔF Change in fluorescence
°C Degrees celsius
2D-GE Two dimensional gel electrophoresis
ABP Antennal binding protein
BAC Bacterial artificial chromosome
BLAST Basic local alignment search tool
bp Base pair(s)
cAMP Cyclic adenosine monophosphate
CCD Charge–coupled device
cDNA Complementary deoxyribonucleic acid
cGMP Cyclic guanosine monophosphate
CMF Chloramphenicol
CSP Chemosensory protein
CT Cycle threshold
C–terminus Carboxy–terminus
DEPC Diethyl pyrocarbonate
DIG Digoxigenin
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide triphosphate
dsDNA Double stranded deoxyribonucleic acid
DSN Duplex-specific nuclease
EAG Electroantennogram
EC50
Half maximal effective concentration
EDTA Ethylenediamine tetra-acetic acid
EST Expressed sequence tag
FID Flame ionisation detector
G Gravitational constant
GC Gas chromatography
xii
List of Abbreviations

List of Abbreviations
gDNA Genomic deoxyribonucleic acid
GOBP General odorant binding protein
GPCR G protein–coupled receptor
G protein Guanine nucleotide–binding proteins
HCl Hydrochloric acid
HEK293 Human embryonic kidney–293
IPM Integrated pest management
IPTG Isopropyl-beta-D-1-thiogalactopyranoside
kb kilo base
kDa kilo Daltons
LB Luria broth
LDS Lithium dodecyl sulphate
mRNA Messenger ribonucleic acid
MWCO Molecular weight cut-off
mtDNA Mitochondrial deoxyribonucleic acid
NBT/BCIP Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl
phosphate
NCBI National centre for biotechnology information
N–terminus Amino–terminus
OBP Odorant binding protein
OD Optical density
ODE Odorant degrading enzyme
OR Olfactory receptor
ORF Open reading frame
ORN Olfactory receptor neuron
OSN Olfactory sensory neuron
PAGE Polyacrylamide electrophoresis
PBAN Pheromone biosynthesis activating neuropeptide
PBP Pheromone binding protein
PBS Phosphate–buffered saline
PCR Polymerase chain reaction
PDE Pheromone degrading enzyme
xiii

List of Abbreviations
pH Potential of hydrogen
PIPES Piperazine-N-N‟-bis(2-ethanesulfonic acid)
PR Pheromone receptor
psi Pound per square inch
PVDF Polyvinylidene fluoride
qRT-PCR Quantitative real time polymerase chain reaction
RACE Rapid amplification of cDNA ends
ROI Region of interest
RNA Ribonucleic acid
rpm Revolutions per minute
S2 Schneider 2
SDS Sodium dodecyl sulphate
Sf9 Spodoptera frugiperda 9
SNMP Sensory neuron membrane protein
ssDNA Single stranded deoxyribonucleic acid
TB Terrific broth
TEV Tobacco etch virus
TM Transmembrane
UTR Untranslated region
UV Ultra Violet
VOBA Volatiles odorant binding assay
xiv

1.1 General overview
1
General Introduction
Olfaction is the ability of organisms to detect and discriminate volatile compounds
from the vast range of odours present in the environment. In animals, this sensory
ability is used for such behaviours as to find a mate, locate food sources and detect
enemies. Chemical cues are used extensively in animals as a means of communication
both within species (intraspecific) and between species (interspecific) (Hartlieb and
Anderson, 1999). Intraspecific chemical signals or pheromones are released by
members of the same species and can be recognised as either releasers or primers.
Releasers are pheromones that take effect immediately, for example, as in kin
recognition, mating stimulation, acting as alarms against dangers, such as predators by
eliciting aggregation, influencing oviposition, territory, trail, recruitment of new
workers, nest building, and leading to food sources. Primers are pheromones that
cause changes in development of the organism, for example, sexual maturation,
development and physiological state (Howse, 1998). Interspecific chemical signals,
which are released by one species and detected by another, can either be a kairomone
whereby the signal benefits the receiver or an allomone, which is beneficial to the
sender, or both. Examples of kairomones include pheromones, toxins, and
metabolites, used in host/prey location and floral scents (of host plants and food
source). Allomones include defence secretions, repellents and floral scents (Jones,
1998).

General Introduction 2
1.2 Olfaction in insects
The olfactory system is the most widely used sensory detection method in insects and
as such is highly specific and receptive to chemical cues in the environment
(Hildebrand and Shepherd, 1997). In insects this highly developed system has
warranted a great deal of focus from research groups and has become a model system
for olfactory studies in general due to the relative small size of the insect olfactory
system compared with higher organisms, the organisation of olfactory receptor
neurons (ORN) into sensilla that has enabled single neuron recordings and the ability
to utilise the knowledge directly in the management of horticultural and agricultural
pests.
1.3 Olfaction in moths
Moths, belong to the order Lepidoptera and are some of the most important pests of
fruit and vegetable crops around the world. The detection of plant volatiles and sex
pheromones by moths has been the major focus of study for the development of pest
control strategies. Much focus has been on sex pheromones and their application with
some other odorous signals or semiochemicals.
1.3.1 Sex pheromones
The first pheromones were first described in moths with the isolation of N-acetyl
tyramine (from cocoons) and bombykol (female sex pheromone), both from the
silkmoth, Bombyx mori (Butenandt et al., 1959a; Butenandt et al., 1959b). Since then
thousands of sex pheromonal compounds have been identified from hundreds of
insect species, as seen in the online pheromone database, (El-Sayed, 2008). Sex
pheromones are produced by either the male or female or both sexes and are the most
widely characterised of all pheromones. Long range sex pheromones are species-
specific volatiles released from the pheromone glands found between the eighth and
ninth abdominal segments of female moths (Tamaki, 1985). The importance of sex
pheromones is in mate location and courtship, where they act as chemical signals to
conspecific males that the female is ready to mate and acts as a guide for the male

General Introduction 3
moth in locating the „calling‟ female over long distances. Lepidopteran sex
pheromones are mostly composed of blends of two or more compounds (major and
minor pheromone components) in specific ratios (Butenandt et al., 1961; Arn et al.,
1986). The ratios are species-specific and the pheromone may fail to elicit a response
in males if altered. Sex pheromones are mainly composed of C10 to C18 unsaturated,
straight chain hydrocarbons together with an oxygenated functional group, for
instance an alcohol, acetate ester or aldehyde (Arn et al., 1992; Jurenka, 2003). Sex
pheromone components in Lepidoptera are synthesised from fatty acids via enzymes
such as acetyl-CoA, followed by desaturation whereby double bonds are placed at
specific positions along the carbon chain. Limited chain shortening reactions then
shorten the carbon chain to the appropriate length and finally reductive modification
of the carbonyl carbon generates the functional group (Foster and Roelofs, 1987;
Foster and Dugdale, 1988; Jurenka, 2003).
1.3.2 Other odorants detected by moths
The extreme sensitivity and specificity of sex pheromone reception has made it the
ideal model for study of olfaction in moths. However, other odorants such as plant
volatiles including esters, aldehydes, alcohols, terpenes and carbon dioxide are also
detected by the moth‟s olfactory system (Kaissling, 1971). These odorants are used by
moths to locate host plants, oviposition sites and food sources. For example, linalool,
a plant volatile, is suggested to be used by B. mori, Heliothis virescens, Helicoverpa
armigera and Spodoptera littoralis in host plant location for laying eggs (Heinbockel
and Kaissling, 1996; Angioy et al., 2003; Røstelien et al., 2005; Anderson et al.,
2009). Herbivore-induced volatiles, which are released by plants in response to insect
feeding, result in the recruitment of beneficial insects that parasitise the infesting
insects. One such example is methyl salicylate, which is detected by moths such as
Mamestra brassica, S. littoralis, and Epiphyas postvittana as a warning that a plant is
already occupied and thus females avoid such plants for laying eggs (Suckling et al.,
1996; Jönsson and Anderson, 1999; Ulland et al., 2008). This is a survival mechanism
used by the moths in order to minimise competition once the larvae hatch. Eugenol,
geraniol and citral have also been shown to act as oviposition deterrents for E.
postvittana, while hexanal, linalool, nonanol, octanol and nonanal act as attractants.
Plant volatiles such as citral, nonanol, octanol and n-decylaldehyde have been shown

General Introduction 4
in behavioural assays to decrease the proportion of E. postvittana laying eggs while
some compounds have implications in decreasing the number of fertile eggs laid per
female moth (Suckling et al., 1996).
1.4 The olfactory system
Antennae are the major odorant detection appendages in moths. Hair-like projections
on the surface of the antennae called sensilla house chemosensory organs that detect
odorants present in the environment (Schneider, 1964). A sensillum has a porous
outer wall and the inner layer is filled with an aqueous fluid, the sensillum lymph
(Breer, 1997). The bipolar olfactory receptor neuron projects a dendritic membrane
into the sensillum lymph, and the other end forms an axon projecting to the antennal
lobe (Keil, 1999). The ORN cell is surrounded by three auxiliary cells, the tormogen,
trichogen and thechogen (Steinbrecht et al., 1992). These cells function in sensillum
structural development and in maintaining the sensillum lymph. Located within the
sensillum lymph are soluble odorant binding proteins (OBPs) and odorant degrading
enzymes (ODEs) and located within the dendritic membrane are the odorant receptors
(ORs) and pheromone receptors (PRs), as depicted in Figure 1.1 (Vogt and Riddiford,
1981; Steinbrecht, 1999; Vogt et al., 2005).

General Introduction 5
Figure 1.1: Structure of a single insect olfactory sensillum (adapted and modified
from Vogt (1987)). Odorants enter the sensillum lymph through pores in the cuticular
wall and interact with the binding proteins present in the lymph which confer them
onto the odorant receptors present on the dendritic membrane of the bipolar olfactory
receptor neuron. Degrading enzymes present in the sensillum lymph may help in the
clearing of odorants from the lymph.
The sensillum types present on moth antennae can be classified into six different
groups. These are the sensilla trichodea, sensilla basiconica, sensilla auricillica,
sensilla chaetica, sensilla styloconica and sensilla coeloconica. Sensilla trichodea are
very long, thin, porous hair-like structures with sharp pointed tips (Schneider, 1964).
Unbranched dendrites of up to three neurons innervate this sensilla type (Keil, 1999).
They are present in high numbers in male moths, while they occur in low numbers or
not at all in female moths and thus were implicated to be involved in recognition of
sex pheromone components released by female moths (Seabrook, 1978). The specific
tuning of this sensilla type to sex pheromones was confirmed by single sensillum
electroantennogram (EAG) recordings (Shields and Hildebrand, 2001). The females
of some moth species also carry large numbers of this sensilla type, however, these
are shorter and the neurons are responsive to general odorants (Nagy and George,
1981). Sensilla basiconica are shorter with blunt rounded tips and are innervated by
the dendrites of up to 50 neurons (Schneider, 1964). This sensilla type has been
implicated in the detection of plant volatiles in S. littoralis (Anderson et al., 1995) and

General Introduction 6
Cactoblastic cactorum (Pophof et al., 2005) in electrophysiological studies. Sensilla
auricillica is a shoehorn shaped, porous sensilla that is innervated by the branched
dendrites of three neurons. They are involved in detecting plant volatiles (Anderson et
al., 2000) and sex pheromone components in Cydia pomonella (Ebbinghaus et al.,
1998; Ansebo et al., 2005). Sensilla chaetica is similar to the trichoid sensilla except
that it has thicker walls and the base has a flexible circular membrane (Schneider,
1964). This sensilla type are either devoid of cuticular pores with a function in
mechano-reception, or have only one pore at their tip and function in gustatory
reception (Keil, 1999). Sensilla styloconica are short, devoid of pores with a peg-like
shape and function as thermo– or hygro– receptors (Shields and Hildebrand, 2001).
The last sensilla type, sensilla coeloconica are very short, double walled, shaped like
peg and are located in pits below the antennae cuticle. The sensilla are aporous but the
double wall is not fused hence odours can move through it. This sensilla type is
innervated by dendrites of five neurons (Altner et al., 1977) and are involved in
detection of aliphatic acids and aldehydes in B. mori and C. cactorum (Pophof, 1997;
Pophof et al., 2005).
The cascade of events that leads from the detection of an odorant present in the
environment and its conversion into a neuronal signal in the antennal lobe of the moth
is still unclear. A proposed mechanism of the perireceptor events that follow detection
of an odorant is as follows (Kaissling, 2009). The hydrophobic odorant molecule
enters the sensillum lymph via waxed pores in the cuticular wall of the sensilla. This
hydrophobic molecule has to travel through the aqueous layer to the membrane bound
receptors in order for signal transduction to occur. Soluble proteins present in
abundance in the lymph may function in transporting the odorants by forming
complexes. The binding of the odorant to the membrane receptor activates a series of
signalling cascades in the ORN causing action potentials to be generated. This results
in the conveyance of the signal to the antennal lobe and processing in higher brain
centres. Odorants need to be cleared from the olfactory system once the signal has
been relayed to the ORNs. This ensures the sensitivity and specificity of the system
and the detection of new odorants or different concentrations of odorant plumes that
direct the moths‟ flight (towards or away from attractants and repellents) (Kaissling,
1974; Vogt, 1987; Prestwich et al., 1989; Kaissling, 2009).

General Introduction 7
1.5 Components of the olfactory system
The identification of several protein components of the olfactory system in insects
such as fruit flies and moths has paved the way for further detailed study of these
proteins and the peripheral events involved in signal transduction. The identification,
isolation and functional analysis of these proteins are described below.
1.5.1 Proteins found in the sensillum lymph
Abundant in the sensillum lymph are small, soluble proteins, ranging in size from 14–
16 kDa called odorant binding proteins (OBP). These proteins are produced in the
sensillum support cells and secreted into the lymph (Vogt and Riddiford, 1981). The
first OBP to be identified was a 15 kDa male antennal specific protein, unique to the
pheromone sensitive sensilla, that bound sex pheromones in the wild silkmoth
Antheraea polyphemus (Vogt and Riddiford, 1981). Due to its close association with
sex pheromones, it was called a pheromone binding protein (PBP). PBPs are generally
found at high concentrations (10 mM) in male insect antennae (Klein, 1987), but
some PBPs have also been identified in female moth antennas (Callahan et al., 2000).
Low levels of PBP have been identified in some Lepidoptera species while high
expression levels of PBPs have been shown in members of the Noctuoidea (Klein,
1987; Callahan et al., 2000). It was not until 1991 when the first evidence of
multigene families of OBPs emerged with the identification of two groups of general
odorant binding proteins (GOBP), namely GOBP1 and GOBP2; proteins that are not
sex-biased and have highly conserved regions within members of the groups (Vogt et
al., 1991; Krieger et al., 1993; Krieger et al., 1996). Most PBPs were identified and
characterised by their binding to radio-labelled versions of known sex pheromone
components. GOBPs on the other hand have been less studied in terms of their
binding partners. In 1997, Feng and Prestwich deduced the ligand of GOBP2 from
Manduca sexta by competitive displacement of tritium-labelled diazoacetate
pheromone analog, [ 3 H]-6E,11Z-16:Dza, by the plant odorants (Z)-3-hexen-1-ol,
geraniol, geranyl acetate and limonene (Feng and Prestwich, 1997). In a recent study
of OBPs from B. mori, GOBP2 was shown to bind the sex pheromone of B. mori,
bombykol and also to be able to discriminate it from its antagonist, bombykal (Zhou
et al., 2009). The specific function of OBPs is still unclear but several roles of OBPs

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

General Introduction 10
opposed to wild type flies that avoid high alcohol levels. Further research with LUSH
mutants showed that this OBP is involved in activity of the pheromone-sensitive
neurons. The activity of the male-specific lipid, 11-cis vaccenyl acetate (cVA) that
causes aggregation in wild type flies is lost in mutants. The activity of this pheromone
is restored with transgenic expression of the LUSH protein suggesting that the loss of
function in mutants is specifically due to absence of LUSH in the pheromone
sensitive sensilla (Zhou et al., 2004; Xu et al., 2005). Further to this, Laughlin et al.
(2008) showed through solving the structures of various bound and unbound states of
LUSH, as well as of several LUSH proteins with point mutations that the cVA bound
LUSH acts as an activator of pheromone sensitive neurons (Laughlin et al., 2008).
When cVA is bound to LUSH, a conformational change occurs that disrupts a salt
bridge in LUSH and causes shifts in its surface loop. This altered state of LUSH then
activates the pheromone neuron, as depicted in Figure 1.2. The salt bridge is
maintained in the unbound state and when an alcohol such as butanol is bound to
LUSH. This indicates that the OBP/ligand complex interacts with the PR (Laughlin et
al., 2008).
Figure 1.2: Odorant receptor activation as depicted by Laughlin et al. (2008). The
formation of the cVA/LUSH complex results in a conformational change of LUSH
making it an active ligand that interacts directly with the pheromone receptor. From
Stowers and Logan (2008).
PR

General Introduction 11
A model has been proposed for ligand deactivation by modification of the PBP in the
pheromone complex to a scavenger form (Kaissling, 2009). In B. mori, when the
PBP/bombykol complex interacts with the receptor molecule on the dendrite of the
ORN, the low pH at the dendrite leads to a conformational change and formation of a
C- terminal α-helix in the PBP, thereby releasing the bombykol (Leal et al., 2005).
Several mechanisms by which the odorant is conferred to the ORs by the
OBP/odorant complex have been proposed. The stable OBP/odorant complex
interacts directly with the OR; or the OBP/odorant complex is unstable and at the
dendritic membrane, the odorant binds to the OR selectively. It could also be that a
dendritic membrane bound docking protein, for example, sensory neuron membrane
protein 1, binds the OBP/odorant complex and this in turn releases the odorant from
the complex and confers it to the OR (Vogt et al., 1985; Prestwich et al., 1995;
Steinbrecht, 1996; Kaissling, 1998). A recent structural based study of A. polyphemus
PBP proposes a pH induced release of odorants from the complex (Zubkov et al.,
2005). Binding of odorant to PBP occurs at neutral lymph pH and release of the
odorant from the complex is achieved by the opening of the ligand binding cavity at
the lower pH near the dendritic membrane.
Other classes of OBPs include chemosensory proteins (CSPs) and antennal binding
proteins (ABPs). The ABPs are expressed specifically in the antennae (Steinbrecht et
al., 1995; Zhang et al., 2001). The CSPs bind odorants and pheromones and also have
implications in taste reception. The first CSP was identified by subtractive
hybridisation experiments in Drosophila melanogaster antennae (McKenna et al.,
1994; Pikielny et al., 1994). Refer to Vogt et al. (2003) for a review.
Another class of soluble proteins present in the sensillum lymph are the odorant
degrading enzymes (ODEs) (Vogt and Riddiford, 1981). Once the signal has been
relayed to the neuron, the odorant needs to be cleared from the lymph so that
sensitivity of the olfactory system is maintained and new signals can be detected as
continuous firing of the neurons with the same odorant can cause a loss of sensitivity
to that odorant. Also, not all odorants that enter the sensillum are detected by the
olfactory system and need to be cleared from the lymph. This may include odorants
toxic to the cells so degradation of these odorants is important in the lymph. This role
in insects is suggested to be played by ODEs such as carboxylesterases, glutathione-

General Introduction 12
S-transferases, and cytochrome P450. See Vogt et al. (1985) and Vogt et al. (2003)
for reviews.
1.5.2 Membrane proteins
Two classes of membrane proteins have been postulated to be of importance to moth
olfaction, the first one being sensory neuron membrane proteins (SNMPs) and the
other being odorant receptors. SNMPs were discovered in 1988 (Vogt et al., 1988;
Rogers et al., 2001) in the antennal lymph of A. polyphemus. There are two classes of
SNMPs (SNMP1 and SNMP2) in moths, with 25 to 75% amino acid identity between
them (Vogt et al., 2003). They were thought to be candidates for the PR of moths due
to their antennal specific expression, pheromone binding ability and expression onset
synchronised with olfactory function development in moths (Vogt et al., 1988; Rogers
et al., 2001a). This hypothesis was later rejected when evidence emerged that SNMPs
were expressed in similar levels in both male and female antennae, had only two
transmembrane domains, were found in most olfactory sensilla types and had low
diversity (Rogers et al., 1997; Rogers et al., 2001). SNMPs are similar to mammalian
membrane bound CD36 proteins. CD36 has implications in cell-to-cell interaction, in
the transport of small hydrophobic molecules and binding protein/lipid complexes
(Vogt et al., 2003). A study carried out by Benton et al. (2007) showed that SNMP is
essential in pheromone induced response of PRs in D. melanogaster (Figure 1.3).
SNMP is expressed in high concentrations in the trichoid sensilla of Drosophila but
does not affect the development of trichoid OSNs and support cells. Several different
pheromone binding experiments showed that Drosophila SNMP is essential for cVA
mediated response of OR67d. H. virescens PR, HvOR13 when expressed in OR67d
neurons also required SNMP to elicit response to the H. virescens pheromone
component (Z)-11-hexadecenal. This study indicates a role of insect SNMPs as a co-
receptor for pheromone reception, due to their preference for binding lipids (Benton et
al., 2007). SNMP may function in olfactory perception of pheromones by interacting
with different components of the olfactory system. It could be involved in
destabilising the PBP/pheromone complex and acting as a preliminary receptor for the
pheromone, conveying it to the PR. It could also couple to the signal transduction
pathway or be involved in signal termination (Rogers et al., 2001; Vogt et al., 2003).

General Introduction 13
Figure 1.3: Model of insect pheromone detection demonstrating a role of SNMP in
cVA mediated response of OR67d, as depicted by Benton et al. (2007). The SNMP
may be acting as a preliminary receptor, destabilising the OBP/pheromone complex
and conveying the pheromone to the PR for signal transduction to occur.
Odorant receptors are seven transmembrane (7TM) domain proteins localised on the
dendritic membranes of ORNs and detect odorants that enter the sensillum lymph
(Benton, 2006). ORs function in binding odorants as they are transported to the
dendritic membrane. This interaction sets off a signalling cascade causing the ORN to
depolarise and a signal is relayed via the axonal end of the ORN to the antennal lobe
in the brain. In contrast to the broadly tuned ORs, pheromone receptors in insects, like
moths, are thought to be highly tuned to sex pheromones (Nakagawa et al., 2005;
Mitsuno et al., 2008; Forstner et al., 2009; Wang et al., 2010). This represents the
holy grail of the insect olfactory system in that the identification of PRs will give a
better understanding of the underlying mechanisms of sex pheromone reception and
pheromone controlled behaviours such as mating.
1.5.2.1 Odorant receptor identification in moths
The first putative ORs to be identified in moths were from the tobacco budworm H.
virescens using the basic local alignment search tool (BLAST) of Heliothis genomic

General Introduction 14
sequence with candidate OR sequences from Drosophila as queries (Altschul et al.,
1990; Krieger et al., 2002). The genomic sequences obtained from the BLAST search
were used as probes for obtaining the exon regions of the sequences from a H.
virescens cDNA library. This process identified nine OR genes in H. virescens and
further screening of the genomic sequences as well as transcriptome sequencing have
led to the identification of a total of 18 ORs in H. virescens so far (Krieger et al.,
2004). Five of these ORs (HvOR11, HvOR13–HvOR16) have been identified as
potential PRs of H. virescens based on the high levels of expression of these genes in
male than female antennae (Krieger et al., 2004; Vásquez et al., 2010). In situ
hybridisations of HvOR11, HvOR13, HvOR14 and HvOR16 showed their expression
was confined to antennal cells below the sensilla trichodea (Große-Wilde et al., 2007;
Baker, 2009; Krieger et al., 2009) and functional characterisation in Xenopus laevis
oocytes has confirmed HvOR13, HvOR14 and HvOR16 to bind (Z)-11-hexadecenal,
(Z)-11-hexadecenyl acetate and (Z)-11-hexadecen-1-ol respectively (Wang et al.,
2010). (Z)-9-tetradecenal forms 5% of the sex pheromone component of H. virescens
and HvOR6, which is expressed in both male and female antennae, and was identified
as the receptor for this pheromone component by expression and characterisation in X.
laevis oocytes (Wang et al., 2010).
The H. virescens ORs share very little homology to other known insect ORs, except
for HvOR2, which is highly conserved across insects and has a role as a co-receptor in
olfactory signalling (refer to section 1.5.2.2 for details of this receptor type).
Digoxigenin (DIG) labelled probes designed to HvOR2 were used to isolate the first
ORs from B. mori and Antheraea pernyi antennal cDNA libraries (Krieger et al.,
2003). Screening of a B. mori male antennal cDNA library as well as the B. mori
genome sequences in GenBank with other H. virescens OR sequences led to the
identification of a total of six ORs (Sakurai et al., 2004; Krieger et al., 2005). In situ
hybridisations has revealed BmOR1 and BmOR3 to be expressed in the sensilla
trichodea, while BmOR2 has dispersed expression not confined to the sensilla
trichodea, and BmOR4 and BmOR5 are expressed in fewer cells. BmOR6 did not
label any cells. Heterologous expression of BmOR1 and BmOR3 in X. laevis oocytes
have identified their ligands as bombykol and bombykal, respectively (Nakagawa et
al., 2005). The availability of the B. mori genome sequence has facilitated the
identification of a total of 68 putative ORs (Sakurai et al., 2004; Wanner et al., 2007;

General Introduction 15
Tanaka et al., 2009), and tissue expression analysis has identified four ORs
(BmOR19, BmOR30, BmOR45 and BmOR47) with higher expression levels in
female than male antennae (Anderson et al., 2009). In situ hybridisation has shown
BmOR19 to be co-localised with either BmOR45 or BmOR47. Heterologous
expression of three of these ORs in Sf9 insect cells has identified BmOR19 to be
tuned to linalool, and BmOR45 and BmOR47 to be tuned to benzoic acid, 2-
phenylethanol and benzaldehyde (Anderson et al., 2009).
Mitsuno et al. (2008) identified ten ORs from three different moth species; Plutella
xylostella (PxOR1, PxOR2, PxOR3 and PxOR4), Mythimna separate (MsOR1,
MsOR2 and MsOR3), and Diaphania indica (DiOR1, DiOR2 and DiOR3), through
degenerate PCR of male antennal cDNA of the moths with primers designed from the
conserved regions of BmOR1 and other male-specific ORs. Three of the ORs were
homologues of the highly conserved BmOR2. Five of the ORs (PxOR1, PxOR4,
DiOR1, MsOR1 and MsOR3) were expressed exclusively in male antennae, while
two (PxOR3, DiOR3) were expressed in both male and female antennae, although
higher levels were observed in male antennae. PxOR1, MsOR1 and DiOR1 were
exclusively expressed in male antennae and shown to bind sex pheromone
component(s) of the respective moths (P. xylostella binds (Z)-11-hexadecenal; M.
separate binds (Z)-11-hexadecenyl acetate and D. indica binds (E)-11-Hexadecenal)
(Mitsuno et al., 2008).
M. sexta ORs were identified by differential screening of a male antennal cDNA
library (OR1), degenerate PCR (OR2, which is the homologue of BmOR2) and from a
subtracted male antennal transcriptome (OR3) (Patch et al., 2009). Tissue expression
analysis revealed MsexOR1 to be expressed in male antennae and MsexOR3 to be
expressed highly in female antennae. Three further ORs, named MsexOR4, MsexOR5
and MsexOR6 have been identified in M. sexta recently from a subtractive male
antennal cDNA library (Große-Wilde et al., 2010). MsexOR4 has been shown to be
expressed in male antennae while MsexOR5 and MsexOR6 have been detected in
female antennae. Functional analysis of MsexOR1 with pheromone components did
not reveal any ligands for this receptor as yet and MsexOR4 has been postulated to
bind one of the sex pheromone components of M. sexta however, no functional data
for odorant binding of these ORs are available as yet.

General Introduction 16
Ten ORs have been identified by degenerate PCR of male moth antennae from eight
different Ostrinia species. The B.mori homologues of BmOR1 and BmOR2 have been
identified from O. scapulalis and O. latipennis, while only the BmOR1 homologue
has been identified in O. furnacalis, O. palustralis, O. zaguliaevi, O. zealis, O.
ovalipennis and O. nubilalis. A further five ORs (BmOR2 homologue and four more
candidate PRs named OnOr3–6) have been identified from a male antennae EST
library of O. nubilalis, bringing the total number of Ostrinia ORs identified to date to
15 (Wanner et al., 2010). Functional analysis of O. scapulalis OR1 (OscaOR1) and O.
latipennis OR1 (OlatOR1) in X. laevis oocytes revealed them to bind (E)-11-
tetradecenol, a sex pheromone component of O. latipennis (Miura et al., 2009). O.
nubulalis OR6 binds Z11-tetradecenyl acetate, a sex pheromone component of
Ostrinia species, while OnOr1, 3 and 5 bind four sex pheromonal components (Z11-
and E11- tetradecenyl acetate, and Z12- and E12- tetradecenyl acetate) and the
behavioural antagonist Z9-tetradecenyl acetate of Ostrinia species (Wanner et al.,
2010).
In the giant silkmoth, A. polyphemus, a candidate PR (ApolOR1) has been identified
from the screening of a cDNA library (Forstner et al., 2009). ApolOR1 is male
antennae specific in its expression and found only in sensilla trichodea. Functional
characterisation showed that it bound the pheromone components of A. polyphemus,
(E,Z)-6,11-hexadecadienyl acetate, (E,Z)-6,11-hexadecadienal and (E,Z)-4,9-
tetradecadienyl acetate (Meng et al., 1989; Forstner et al., 2009). A summary of the
moth PRs and the sex pheromone component each binds is given in Table 1.1.

General Introduction 17
Table 1.1: Summary of the moth pheromone receptors identified and characterised to
date, together with their sex pheromone binding components. Where more than one
sex pheromone component is given, the component marked with * is the pheromone
component bound by the corresponding PR. O. nubulalis OR1, 3 and 5 bind all the
four corresponding pheromone components given.
Moth and
Reference
study
H. virescens
(Vetter and
Baker, 1983)
B. mori
(Kaissling and
Kasang, 1978)
A.polyphemus
(Meng et al.,
1989)
P. xylostella
(Mitsuno et
al., 2008)
D. indica
(Mitsuno et
al., 2008)
M. separate
(Mitsuno et
al., 2008)
O. scapulalis
(Miura et al.,
2009)
O. latipennis
(Miura et al.,
2009)
O. nubulalis OR1
OR3
OR5
PR Sex pheromone component
bound by corresponding PR
Percentage of
pheromone blend
component
HvOR6 (Z)-9-tetradecenal 5
HvOR13 (Z)-11-hexadecenal 73
HvOR14 (Z)-11-hexadecenyl acetate Produced by H. subflexa
HvOR16 (Z)-11-hexadecen-1-ol 1
BmOR1 Bombykol 90
BmOR3 Bombykal 10
ApolOR1 *(E,Z)-6,11-hexadecadienyl
acetate
90
(E,Z)-6,11-hexadecadienal 10
(E,Z)-4,9-tetradecadienyl acetate Trace amounts
PxOR1 *(Z)-11-hexadecenal 50
(Z)-11-hexadecenyl acetate 50
(Z)-11-hexadecen-1-ol Trace amounts
DiOR1 *(E)-11-Hexadecenal 66.1
(E,E)-10,12-hexadecadienal 32.6
MsOR1 *(Z)-11-hexadecenyl acetate 55.8
(Z)-11-hexadecen-1-ol 29.2
OR1 (E)-11-tetradecenol Not the sex pheromone
for this moth but is for
O. latipennis
OR1 (E)-11-tetradecenol Unknown
Z11- and E11- tetradecenyl
acetate, and Z12- and E12tetradecenyl
acetate
OR6 Z11-tetradecenyl acetate
97:3 (Z11-, Z12- : E11-,
E12)

General Introduction 18
1.5.2.2 Insect odorant receptor activation
The novel receptor type in insects, OR83b is a 7TM domain protein that is found co-
expressed with the OR in almost all ORNs. Even though OR diversity within and
between insect species is high, this receptor type is highly conserved in insects such
as D. melanogaster, B. mori, A. pernyi, H. virescens, Tenebrio molitor, Apis mellifera,
Calliphora erythrocephala, P. xylostella, M. separate, D. indica, and E. postvittana
(Vosshall et al., 1999; Hill et al., 2002; Krieger et al., 2002; Larsson et al., 2004;
Melo et al., 2004; Mitsuno et al., 2008; Jordan et al., 2009). The OR/OR83b
heteromeric complex is formed in the cell body and it is thought that the OR83b
functions in transport of the bound OR to the dendritic membrane in the sensilla. The
complex is maintained in the sensilla; suggesting a role of OR83b as a co-receptor in
olfactory signalling. Supporting evidence for this could be provided by the high
sequence identity of this receptor type across different insect species (64%–88%).
Orthologs of OR83b in other insect species have a conserved co-expression function
and may be an important factor in odorant detection by conventional ORs in insects
(Larsson et al., 2004; Neuhaus et al., 2004; Nakagawa et al., 2005).
The first insect ORs were identified in Drosophila through homology search of the
draft Drosophila genome using novel computer programs that identify mammalian
ORs called G protein-coupled receptor (GPCR) statistically by their physicochemical
properties (Clyne et al., 1999; Vosshall et al., 1999). These algorithms were used to
identify open reading frames of two or more transmembrane domain proteins. Both
GPCRs and insect ORs have 7TM domains, however, insect ORs have been
demonstrated to have an intracellular N-terminus, a topology unique to insect ORs, as
shown in Figure 1.4 (Benton et al., 2006). Lundin et al. (2007) used glycosylation
scanning to show the orientation and confirm the number of TM domains in
Drosophila OR83b. Further evidence for insect OR topology came from epitope
tagging of the termini and the predicted loop regions of Drosophila OR22a (Smart et
al., 2008). This unique orientation of insect ORs suggests that these receptors would
use distinct insect-specific signalling pathways.

General Introduction 19
Figure 1.4: Topologies of insect and mammalian odorant receptors in the cell
membrane showing opposite orientation of the N- and C- termini in insect OR as
opposed to mammalian OR, as depicted by Benton et al. (2006), Lundin et al. (2007)
and Smart et al. (2008).
Recent evidence suggests a novel role of OR83b as an ion channel, that when co-
expressed with a non-OR83b OR, confers ligand sensitivity (response shown by light
blue arrow on Figure 1.5). Sato et al. (2008) heterologously expressed a number of
different insect receptors in HeLa cells, together with the co-receptor OR83b and
measured calcium influx and whole-cell currents to analyse early onset of OR
activation. They showed that the calcium influx response of the insect OR expressing
cells to odorant stimulation is ten-fold faster than for mammalian ORs and no G
proteins are involved in the signalling, therefore none were inhibited, supporting
similar findings by Smart et al. (2008). The response was also independent of second
messengers such a cGMP and cAMP. The ion selectivity which is a property of ion
channels, was dependent on the OR subunit composition; evidence suggesting that
ORs themselves act as ion channels rather than being associated with separate ion
channels (Sato et al., 2008; Smart et al., 2008).
A second hypothesis proposed by Wicher et al. (2008) suggests an ion channel–
GPCR model (Figure 1.5). The ion channel model, similar to Sato et al. (2008)
suggests OR83b forming a channel complex with conventional ORs when co-
expressed in HEK293 cells to evoke odour responses upon ligand binding. The GPCR
model shows that the binding of a ligand to its OR/OR83b complex leads to the

General Introduction 20
opening of a cAMP dependent channel, suggesting the odour evoked response also
activates G proteins. The activation of the OR complex occurs rapidly while the G
protein activation is slower and longer lasting (Wicher et al., 2008). No such evidence
for a metabotropic signalling mechanism has been observed in any other studies and
further experiments have to be conducted to rectify the differences in these three
studies.
Figure 1.5: The ion channel–GPCR model of odorant signalling in Drosophila. The
ion channel formed by OR22a/OR83b complex depicts a rapid, short lived odorant
evoked response upon stimulation by the ligand ethyl butyrate (light blue arrow) as
suggested by Sato et al. (2008), Smart et al. (2008) and Wicher et al. (2008). The
GPCR model suggests a slow and prolonged response upon ligand binding to the
OR22a/OR83b complex, leading to an increase in cAMP production that opens up the
motif that controls ion permeability in OR83b, thus resulting in membrane
depolarisation (green arrow) as suggested by Wicher et al. (2008). This figure has
been adapted from Wicher et al. (2008).
ORs are a highly divergent group in that there is very little sequence homology both
between and within species, for example, D. melanogaster ORs share only 17%–26%
amino acid sequence identity (Vosshall et al., 2000); and the moth-specific
pheromone receptor clade has an overall amino acid sequence identity of 10% (Jordan
et al., 2009). They are broadly tuned as one OR can bind many odorants with different
specificities and different ORs can bind the same odorants. For example, B. mori
OR45 binds five odorants, three of which are also ligands for BmOR47 (Anderson et
al., 2009). Other studies in insects support this broad tuning of ORs (Malnic et al.,
1999; Touhara et al., 1999; Malnic et al., 2004; Hallem and Carlson, 2006; Anderson

General Introduction 21
et al., 2009; Jordan et al., 2009). The results in these studies show that the expression
levels of ORs and PRs not only vary between the sexes but different ORs are
expressed at different levels within a specific tissue. A combinatorial system is used
by tens to hundreds of ORs to clearly detect and discriminate hundreds to thousands
of different odorants. This combinatorial functioning of ORs can be attributed to the
organisation of the olfactory machinery, as different ORNs expressing the same ORs
converge to the same glomerulus in the antennal lobe of the brain (Vosshall et al.,
2000; Couto et al., 2005; Fishilevich and Vosshall, 2005).
1.6 Insect pest control strategies
The problem of insect pests has been ongoing for centuries. They cause damage to
food and crops in the field and in storage, and therefore measures need to be taken to
bring insect pests under control and minimise the damage and hence loss to the plant-
based industries. Current insect pest control strategies include spraying food crops
with pesticides, transgenic plants, as well as olfaction-based methods. These methods
include lures to monitor pests in fields, mass trapping of insects and killing them (lure
and kill approach) and the use of mating disruption and use of biological controls
(Jones, 1998; Suckling and Karg, 1999; Suckling et al., 1999).
Control strategies using pheromones and plant volatiles have achieved success to
some extent. Mating disruption reduces the number of successful matings and hence
the number of larvae that cause damage to plants (Jones, 1998). This system employs
the release of sex pheromones in the atmosphere that masks the release of pheromone
from calling female insects. This pheromone plume in the atmosphere confuses the
males which are unable to detect the trail of pheromone towards the female thus
reduces the number of successful mating. The success of this strategy is dependent on
a number of factors. Knowledge of the time and place of mating will ensure that the
pheromones are released at the right time and the number of generations of the target
insect in a year will also be indicative of the number of pheromone applications that is
needed. The life expectancy of the target insects is an important factor also in that the
shorter time the adults have, the better the rates of mating disruptions that can be
achieved. The chances of an adult male in locating a female is less if the adult is short

General Introduction 22
lived but it increases if the adult is long lived (as they have more time in locating the
female amidst the pheromone plumes) (Campion et al., 1989). This control method is
sustainable as pheromones are non-toxic, non pollutant, naturally occurring
compounds and can be used without causing harm to human health or the
environment. Since they are species specific, they do not affect other non-target or
beneficial insects. This is not to say that there are no limitations to this method.
Because pheromones are insect specific and occur in specific ratios of the major and
minor components of the pheromone, the target range is limited (Campion et al.,
1989). Its effectiveness is limited to just one particular species and different blends
have to be synthesized for different target insects. The timing of application has to be
precise for it to be effective. The insect density in the controlled area has to be
monitored. Low density in a large area is preferred as a lower population of mating
adults have less chance of locating mates in larger areas. If the control area has
uncontrolled regions nearby, for example home gardens, then mated females from
these areas can migrate and lay eggs in the controlled region. The larvae from these
eggs will still cause damage to plants in the controlled region. Finally, the cost
effectiveness of such a system is questionable as pheromones are expensive to
synthesise and are unstable volatiles that disintegrate easily in the environment
(Bakke and Lie, 1989; Campion et al., 1989; Wall, 1989).
Mass trapping of insects is achieved by using lures such as sex pheromones and plant
volatiles of host plants to direct insects to traps (Haynes et al., 1986). Once trapped,
the insects are killed, either by toxins in the trap or by depriving them of oxygen. The
traps may also be coated with sticky substances to which the insects get stuck and die.
Mass trapping is done to reduce the population of insects so that less mating pairs are
available and the number of offspring in the new generation is reduced (Bakke and
Lie, 1989). The effectiveness of mass trapping is reduced in highly populated areas
when the traps become full quickly. When sex pheromones are used as the lure, only
male moths are caught, and the males that are not caught can fertilise more than one
female hence a high proportion of the males have to be caught to render this method
effective. Another approach that has been used in the control of insect pests is lure
and kill. A lure, such as sex pheromone that is able to attract insects is associated with
an insecticide that acts to kill the insects. The effectiveness of this system depends on

General Introduction 23
the attractiveness of the lure and the ability of the insecticide to infect and kill the
insect (Haynes et al., 1986).
One approach to overcome the shortfalls of mass trapping and lure and kill is lure and
infect (Suckling and Karg, 1999). This has been used on insects such as tobacco
budworm (Jackson et al., 1992), codling moth (Hrdy et al., 1996) and diamondback
moth (Pell et al., 1993). This approach uses lures such as pheromones combined with
insect pathogens such as virus, bacteria or fungi to attract insects to the lure source
(O‟Callaghan and Jackson., 1993). Once infected, the insects act as vectors of the
disease and spread it across the population. An advantage of using such an approach
is the speed with which the disease spreads in the population and with the insect
themselves acting as vectors for the pathogen, the continuous need for the lure source
to be maintained and the number of lure sources is reduced (Suckling and Karg,
1999). The downfall of this system is that bacterial toxins and viruses are pathogenic
only when consumed hence the lure system has to be designed such that the pathogen
is consumed by the insects. Also, for the pathogen to spread in the population, the
infected males have to mate to pass on the pathogen to the females, which must then
be able to spread it to the surface of the eggs they lay, which will eventually have to
be consumed by the larvae at eclosion. The success of the lure and infect approach
using bacteria or virus as the pathogen hence largely depend on the efficiency by
which these series of events take place. When fungi is used as the pathogen, the lure
has to be presented in such a way that it is able to attract the adult insects and keep
them within the vicinity of the lure long enough for the fungi to be picked up. The
adult has to be able to spread the fungi to other insects in the population before dying
or upon death; the fungi will grow on the dead insect and spread within the
population. However, the fungi might be susceptible to environmental factors such as
ultra violet (UV) radiation and die, a limiting step to the success of this system.
Economic production of the pathogen, maintenance of the pathogen in the
environment, development of an efficient delivery system and ability of the pathogen
to spread across the population are factors that have to be taken into consideration
when designing lure and infect control strategies (Suckling and Karg, 1999).
Other methods of reducing the damage caused to plants are spraying the host plants
with feeding and ovipostion deterrents. Oviposition deterrents will prevent females

General Introduction 24
from laying eggs on host plants and the feeding deterrents will render the plant
inedible. The best approach as yet is the integrated pest management (IPM) system
which was developed to increase the use of biological resources (biological control
and mating disruption) and supplement its use with reduced amounts of chemicals
such as pesticides (Wearing, 1993; Jones, 1998).
1.7 Light brown apple moth
The light brown apple moth, Epiphyas postvittana, is a species of leafroller moth, a
member of the order Lepidoptera and belongs to the family Tortricidae (Wearing et
al., 1991). E. postvittana is endemic to Australia but has also invaded Hawaii,
California and parts of Europe (Suckling and Brockerhoff, 2010). The first report of
this moth in New Zealand was in 1891 (Danthanarayana, 1975). E. postvittana has a
wide host range, from pipfruit (apples and pears) to citrus, grapes, kiwifruit and even
some vegetables. E. postvittana attack pipfruit in all the fruit growing regions of New
Zealand and is found in very high numbers in Nelson (Wearing et al., 1991). The
moth does not have a winter dormancy stage and the number of generations in a year
differs in different regions of the country, with the far north having four generations,
central parts of the country having three and two generations in the southern region. If
the optimum temperature for larval development, 20°C is provided, it will take
approximately 32–40 days for the completion of all the larval developmental stages.
The larvae feed on leaves and fruits by creating silken shelters on either a single leaf
by rolling or between a leaf and fruit (Geier and Briese, 1980). Pupae turn from a
green to brown colour with maturation and male moth emergence occurs before
females. The female moth mates only once in her life, and the eggs are laid in several
batches of 30 eggs each so one female moth can lay between 150–900 eggs. Most of
these eggs hatch into larvae as predation of the eggs is very low. Even though some
biological control agents were imported from Australia, the zero tolerance
requirement of this moth on exported fruits has still not been attained (Wearing et al.,
1991).
The damage causing stage of E. postvittana is the larva, which feeds on the buds,
foliage, shoots and fruits (Suckling, 1998; Markwick et al., 2002). The rolling of

General Introduction 25
leaves and webbing of leaves to fruits to make protective shelters causes calluses on
the fruits and leaves (Lo et al., 2000). The export market requires nil tolerance of both
insect and insect damage on fruits. These stringent measures put in place calls for the
development of better and sustainable control measures for this pest. From the 1960s,
chemical control was used in the form of pesticide sprays. The major chemicals used
were azinphos-methyl and chloropyrifos sprayed on fruits. However, these chemicals
are toxic to some natural enemies of the moth and in 1980s, populations resistant to
the chemicals were reported. This called for alternative control measures. Bacillus
thuringiensis (B.t) was looked into but due to high UV radiation in New Zealand, this
microbial insecticide is easily deactivated (Suckling et al., 1994; Suckling et al.,
1999; Markwick et al., 2002). The naturally occurring baculovirus enemy of the moth
that kills the larvae, nucleopolyhedrovirus was used as sprays to some success.
However, the most sustainable control came from the use of mating disruption and
pheromone trapping of the adult moths. Small scale mating disruption showed that the
best results were achieved through integrated pest management (IPM), by using the
20:1 ratio of the sex pheromone components together with a few applications of
insecticide (Suckling and Shaw, 1990). Scale up of this mating disruption showed
similar results in larger fields (Suckling and Shaw, 1995). The efficiency of this
system is however hindered by factors such as migration of mated females from
nearby untreated sources, or a high population density requiring a second application
(McLaren et al., 1998). The biggest issue facing mating disruption is the high
concentration of pheromone required hence the high cost of this pest control method.
The limited knowledge of actual pheromone mechanism hinders the success of this
system. Understanding the underlying mechanisms of receptor adaptation to different
compounds, the actual behaviour that mating disruption targets and whether males
respond to any remaining pheromone plumes will enhance its success. Reviewed in
Suckling and Brockerhoff (2010).
The sex pheromone of E. postvittana is released at night or during the dark period,
and has been identified to be a 20:1 ratio of (E)-11-tetradecenyl acetate (E11-14:OAc)
and (E,E)-9,11-tetradecadienyl acetate (E9,E11-14:OAc), respectively (Bellas et al.,
1983) shown in Figure 1.6. The components on their own do not produce any
response in male moths but when present in the specific ratio, the blend produced
response in both field trials and laboratory bioassay. The biosynthetic pathways

General Introduction 26
involved in the production of the sex pheromone blend have been shown. The
precursor, myristic acid underwent E11 desaturation event, followed by reduction and
acetylation to give the pheromone component E11-14:OAc. E11 desaturation of the
precursor palmitic acid followed by chain shortening yielded E9-14:OAc and the
diene resulted from further E11 desaturation (Foster and Roelofs, 1990). The
pheromone production in female moths is regulated by pheromone biosynthesis
activating neuropeptide (PBAN). The presence of sex pheromone in the female is low
at eclosion, reaches its highest levels two days after eclosion and decreases to one
third that of the peak at day seven, a gradual decline with the age of the moth (Foster,
1993). Mating results in lowering release of PBAN from females and thus stops the
female from producing any more pheromone (Foster and Greenwood, 1997).
Figure 1.6: The sex pheromone blend of E. postvittana is a 20:1 ratio of (E)-11tetradecenyl
acetate and (E,E)-9,11-tetradecadienyl acetate (Bellas et al., 1983).

General Introduction 27
Figure 1.7: Scanning electronmicrograph of adult male (left) and female (right)
antennaes of E.postvittana. The pheromone sensitive long sensilla trichodea are
present in higher numbers on the male antennae than the female (shown with yellow
arrow) suggesting a role in sex pheromone reception of male moths (Jordon, 2006).
Scale bars = 0.1mm.
Pheromone and plant volatile recognition experiments in E. postvittana have been
done using EAG response recordings of antennae and ORNs (Rumbo, 1981; Karg et
al., 1992; Suckling et al., 1996). The long sensilla trichodea localised in two radial
rows on each male antennal segments are sensitive to pheromone components
(Rumbo, 1981). This sensilla type is not present in the female antennae, as shown by
scanning electron microscopy (Figure 1.7). Experiments showed that each sensilla
(including the pheromone sensitive sensilla) had three ORN cells associated with it,
however, only two of these three were shown to be active in the pheromone specific
sensillum (Rumbo, 1983). To determine what specific plant odorants the male and
female moths respond to, EAG experiments and oviposition assays were carried out
(Suckling et al., 1996). The odorants were chosen based on their presence in fruit and
host plants of the moth (Table 1.2). These experiments revealed that eugenol, geraniol
and citral acted as oviposition deterrents while hexanal, linalool, nonanol, and octanol
were attractants for the moth.

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),

General Introduction 30
this receptor is referred to as BmOR49 but in Tanaka et al. (2009), another OR has
been annotated as BmOR49 so in this thesis, the BmOR49 of Anderson et al. (2009)
will be referred to as BmOR49J], and tissue expression analysis showed it to be
expressed in both male and female antennae at similar levels. Functional
characterisation of EpOR3 by heterologous expression in Sf9 cells decoded its ligands
to be members of terpenes, alcohols and esters at high concentrations, while at lower
concentrations, the receptor bound citral, geraniol, geranial and geranyl acetate. Dose
response curves showed EpOR3 has the highest affinity for the oviposition deterrent
citral, with an EC50 value of 1.1 x 10 -13 M (Jordan et al., 2009), implicating a role of
this receptor as an OR and not PR. Three ORs have so far been identified from E.
postvittana; EpOR2 is the Drosophila OR83b homologue, EpOR3 is the receptor for
general plant odorants while EpOR1 remains to be functionally characterised. Further
analysis of these receptors and the identification of new receptors will lead to a better
understanding of the olfactory mechanisms of this moth. This knowledge will aid in
the development of pest control strategies targeting the olfactory system of the moth.
Two GOBP members have also been identified; however this class of soluble proteins
have very little functional data available, therefore an attempt will be made to deduce
the role(s), if any, of this class of protein in odour perception in in vitro assay
systems.

General Introduction 31
BmOr2
HvOr2
MsepOR2
DiOR2
PxOR2
EpOR2
0.1
100
*
80
HvOr15
HvOr14
MsepOR1
HvOr6
HvOr16
HvOr11
BmOr3
PxOR1
BmOr7
BmOr5
BmOr9
BmOr4
MsepOR3
HvOr13
DiOR3
BmOr1
DiOR1
EpOR1
PxOR3
PxOR4
Male-biased
sex pheromone
receptor clade
31
77
99
BmOr6
100 BmOr22
BmOr21
HvOr19
41 62
99
100
98 HvOr21
BmOr19
BmOr17
BmOr8
BmOr20
100
43
BmOr38
BmOr35
BmOr37
96 HvOr12
45
41
BmOr24
69
BmOr25
99
100
88 89 HvOr7
BmOr11
HvOr9
BmOr23
BmOr42
54
100 HvOr18
40
100
100
HvOr20
BmOr28
57
95
BmOr14
100 BmOr34
100
BmOr33
BmOr30
BmOr36
34
HvOr3
100 BmOR22J
100
BmOr15
99
BmOr12
30 73
100
100
HvOr8
BmOr13
EpOR3 *
EpOR3
BmOR49 BmOR49J
100
100 BmOr48
BmOr46 Female-biased
100 BmOr47
BmOr45 receptor clade
100 BmOR19J
94
97
HvOr10
BmOr10
40
100
BmOr16
89 BmOR23J
BmOr26
HvOr17
98
BmOr29
BmOr27
Or83b clade
98
99
48
100
60
25 100
Figure 1.8: Phylogenetic tree constructed from lepidopteran odorant receptors with
the three E. postvittana ORs indicated by *. Adapted from Jordan et al. (2009) where
the tree was originally constructed using the FITCH method from Jones-Thorton
distances and the given bootstrap values calculated using 1000 replicates.
50
62
96
33
100
25
34
91
100
94
100
100
100
*

General Introduction 32
1.8 Aims
The implications of E. postvittana on the agricultural industries of New Zealand,
Australia and California have increased the interest in studying the mechanisms of
olfaction of this pest. In moths, pheromone receptors have been shown to be the
receptors for species-specific sex pheromones and thus may play a crucial role in
male moth successfully perceiving sex pheromones released by calling females.
Odorant receptors on the other hand bind a range of plant volatiles and might be
involved in host plant location in moths. With the crucial role that these proteins play
in the reproduction and survival of moths, a starting point in combating these
agricultural pests would be to identify and deorphan their ORs and PRs. The aims of
this study are to deorphan the binding partners of EpOR1, which is phylogenetically
related to PRs of other moth species; to investigate the role(s) of GOBP2 from E.
postvittana in odorant recognition and to identify novel ORs and the PR(s) from E.
postvittana.
In chapter 2, EpOR1 is functionally characterised to identify its binding partners;
whether it binds the sex pheromone of E. postvittana predicted by its phylogenetic
relatedness to PRs from other moth species or whether it binds semiochemicals of
general importance to the moth due to similar levels of expression in both male and
female antennae. The binding of EpOR1 to its ligands and the relative sensitivity to
different odorants is assessed by estimating EC50 values by obtaining dose response
curves of ligand binding.
In chapter 3, the role of EpGOBP2 in ligand binding is investigated. Current evidence
suggests up to five roles of PBPs in odorant perception of moths, however, few if any
tests have been conducted for the roles for GOBPs. The ability of recombinant
EpGOBP2 to bind the ligands of EpOR1 is tested, and common ligands for these two
proteins are established. The Sf9 cell assay system used for characterising EpOR1 is
reconstituted with EpGOBP2 to test roles in odorant perception, including odorant
solubilisation, transport and selectivity.
In chapter 4 an attempt to increase the receptor repertoire of E. postvittana is made. A
total of 68 ORs have been identified from B. mori so far and this is used as the model

General Introduction 33
for lepidopterans. Four different methods are compared to achieve this goal including
microarray analysis, degenerate PCR, deep transcriptomics and low coverage whole
genome sequencing. Questions on how the E. postvittana receptor repertoire
correlates with that of B. mori receptor repertoire are addressed and tissue expression
analysis of the ORs are conducted to identify any receptors that show sex-bias in their
expression.
Finally the general discussion, synthesising the data obtained in this research with
recent advances in lepidopteran olfaction is presented in chapter 5. A summary of the
E. postvittana odorant receptors identified to date is presented, together with
recommendations for future experiments that will confirm their tissue expression and
deduce their roles in E. postvittana olfaction.

2.1 Introduction
2
Functional
Characterisation of
Epiphyas postvittana
Odorant Receptor 1
Olfaction, or the sense of smell, plays a major role in the survival and reproduction of
moths whereby chemical signals present in their environment are used to
communicate both within and between species. Species-specific sex pheromones are
released in the environment by females ready to mate and this is perceived by male
moths over distances of up to 100 metres. Moths are also able to detect and
discriminate a vast range of plant volatile compounds that guide them to food sources,
inform them of plants already under herbivore attack and guide females to suitable
oviposition sites (Honda, 1995; Bruce et al., 2005).
ORs and PRs have been implicated as major players in odour detection for several
moth species (Nakagawa et al., 2005; Große-Wilde et al., 2007; Mitsuno et al., 2008;
Anderson et al., 2009; Forstner et al., 2009; Jordan et al., 2009; Miura et al., 2009).
Genes encoding moth ORs and PRs have been identified in a number of different

Functional characterisation of Epiphyas postvittana odorant receptor 1 35
species using degenerate PCR, transcriptome sequencing and whole genome
sequencing techniques; E. postvittana (Jordan et al., 2009), A. polyphemus (Forstner
et al., 2009), B. mori (Sakurai et al., 2004; Krieger et al., 2005; Nakagawa et al.,
2005; Wanner et al., 2007; Tanaka et al., 2009), P. xylostella, M. separata, D. indica
(Mitsuno et al., 2008), H. virescens (Krieger et al., 2002; Krieger et al., 2004),
Spodoptera exigua (Xiu et al., 2004) A. pernyi (Krieger et al., 2003), M. sexta (Patch
et al., 2009; Große-Wilde et al., 2010) and eight different Ostrinia species (Miura et
al., 2009; Wanner et al., 2010). The majority of ORs from moths have been identified
in B. mori and H. virescens.
Typically in studies, the usual approach to characterise novel OR genes are to group
them into clades based on their phylogenetic relationships. The expression levels of
the ORs can be then tested in different tissues in male and female moths, those that
show sex biased expression patterns are classified as putative pheromone receptors.
De-orphaning the receptors using available assay systems is carried out to confirm
they are pheromone receptors and identify which components of sex pheromone
blends they bind. Pheromone receptors have so far been identified and functionally
characterised for a number of moth species including B. mori, H. virescens, P.
xylostella, M. separata, D. indica, A. polyphemus, O. scapulalis, O. latipennis, and O.
nubulalis, see section 1.5.2.1 (Nakagawa et al., 2005; Mitsuno et al., 2008; Forstner et
al., 2009; Miura et al., 2009; Wang et al., 2010).
Attempts to decode the olfactory systems of moths have seen the use of EAG to show
the recognition of a vast range of important plant semiochemicals including esters,
terpenes, alcohols, aliphatics and aldehydes by different species of moths. These may
aid the insects in locating oviposition sites and food sources (Suckling et al., 1996;
Suckling et al., 1996; Fraser et al., 2003; Das et al., 2007). This method gives a broad
picture of the wide range of volatiles recognised by the moth, however, information
on the receptive range of individual ORs, which is at the forefront of investigating
into the molecular mechanisms of insect olfaction is lacking. Several characterisation
assay systems have been successfully developed and used to confer functionality to
individual ORs over the years. These include either the in vivo empty-neuron system
developed in Drosophila (Stortkuhl and Kettler, 2001; Dobritsa et al., 2003) or in
vitro assays in heterologous cell-based expression systems (Wetzel et al., 2001;

Functional characterisation of Epiphyas postvittana odorant receptor 1 36
Neuhaus et al., 2004; Sakurai et al., 2004; Nakagawa et al., 2005; Kiely et al., 2007;
Sato et al., 2008; Anderson et al., 2009; Jordan et al., 2009).
The empty-neuron system, a mutant fly strain that does not express the OR22a and
OR22b receptors was originally developed for characterising Drosophila ORs
(Dobritsa et al., 2003).This approach has been successfully applied to the expression
and characterisation of ORs from other insects such as mosquitoes and moths (Syed et
al., 2006; Kurtovic et al., 2007; Lu et al., 2007; Syed et al., 2010). Syed et al. (2006)
successfully co-expressed functional B. mori OR1 with B. mori PBP in the
Drosophila empty-neuron system and showed that it bound the B. mori sex
pheromone, bombykol, albeit with low signal onset that lasted for an unusually long
time, implying the empty-neuron expressing the BmOR1 is devoid of pheromone
degrading enzymes that would otherwise degrade bombykol (Syed et al., 2006).
Functional studies of moth ORs have mainly been carried out using heterologous
expression systems. Several assays for characterising moth ORs have been carried
out in X. laevis oocytes by two-electrode voltage clamping (Sakurai et al., 2004;
Mitsuno et al., 2008; Miura et al., 2009). Briefly, the OR of interest is co-expressed
with the species-specific Drosophila OR83b homologue in X. laevis oocytes and the
cells are left to grow for 2–3 days at 20°C. Two-electrode voltage clamps are used for
recording whole-cell currents which measures response upon stimulation with
odorants as a variation from the holding voltage.
Sakurai et al. (2004) expressed the B. mori pheromone receptor, BmOR1 together
with BmGαq in X. laevis oocytes and showed that 10 to 15% of the transfected cells
were responsive to the B. mori sex pheromone bombykol with an EC50 value of 3.4 x
10 -5 M (Sakurai et al., 2004). In a similar experiment, the B. mori homologue of
OR83b, BmOR2 was co-expressed with BmOR1 and BmGαq in X. laevis oocytes
(Nakagawa et al., 2005). Stimulation of the transfected cells with bombykol rendered
~95% of the transfected cells responsive, with an EC50 value of 1.5 x 10 -6 M and the
lowest threshold concentration for response of 1 x 10 -7 M, indicating a role of OR83b
in enhancing the sensitivity of ORs.

Functional characterisation of Epiphyas postvittana odorant receptor 1 37
Miura et al. (2009) co-expressed O. scapulalis OR1 (OscaOR1) together with its
species-specific OR83b homologue in X. laevis oocytes and showed that it responded
highly to E11-14:OH and a small response was shown to Z11-16:OAc (Miura et al.,
2009). These compounds have been identified as pheromone components of other
Ostrinia species, while the sex pheromone components of O. scapulalis (E11- and
Z11- 14:OAc) did not confer a response from OscaOR1, indicating that even though
OscaOR1 is homologous to the pheromone receptors of Ostrinia species, it is not
specific to O. scapulalis sex pheromone. Wanner et al. (2010) expressed Ostrinia
nubilalis OR1, and ORs 3–6 in X. laevis oocytes and showed that they bound all the
components of the Ostrinia sex pheromone, as shown in Table 1.1 (Wanner et al.,
2010).
Mitsuno et al. (2008) expressed the pheromone receptor together with their species
specific OR83b homologue from three different moth species (P. xylostella, M.
separata and D. indica) in X. laevis oocytes and showed via electrophysiological
recordings that they all bound their respective sex pheromone components with an
EC50 in the micromolar range and the lowest binding threshold of 10 -7 M, values
which are comparable with B. mori PR assays conducted by Nakagawa et al. (2005).
Modified versions of HEK293 cells, containing a mouse Gα15 gene have also been
used for successfully expressing and characterising moth ORs in fluorometric assays.
Stable cell lines of H. virescens HvOR13, HvOR14 and HvOR16 have been generated
and characterised to bind H. virescens sex pheromone components in HEK cells with
high affinity. HvOR13 can recognise Z11-16:Al in DMSO with an EC50 of 1.2 x 10 -9
M (Große-Wilde et al., 2007). These results are comparable to an earlier study in
which B. mori OR1 and OR3 were expressed and characterised in HEK293 cells.
BmOR1 bound the sex pheromone bombykol with an EC50 of 10 -10 M (Große-Wilde
et al., 2006). Together these results show that expression of moth ORs in HEK293
cells yield functional ORs, that are highly responsive and sensitive to their ligands.
Kiely et al. (2006) developed an insect cell system for assaying insect ORs.
Drosophila OR22a was transiently expressed in Sf9 cells, and after a 48 hour
incubation period, the cells were loaded with the calcium sensitive dye Fluo-4 and
change in fluorescence of cells in response to stimulation by odorants was measured

Functional characterisation of Epiphyas postvittana odorant receptor 1 38
in a Leitz Fluovert FS inverted microscope (Kiely et al., 2007). OR22a was shown to
respond to ethyl butyrate and other odorants previously characterised by in vivo
assays (Dobritsa et al., 2003; Hallem et al., 2004; Hallem and Carlson, 2006). Using
insect cell lines would provide the best conditions for assaying insect ORs as the
heterologously expressed ORs are able to efficiently couple with the Sf9 cell signal
transduction pathway. Evidence for this can be seen in the threshold concentrations of
ethyl butyrate that is required to elicit a response in OR22a. When OR22a is
expressed in HEK293 cells, a high concentration of 10 -3 M of ethyl butyrate is
required to elicit a response (Neuhaus et al., 2004). This threshold concentration is
decreased by 10 8 -folds to 10 -12 M when OR22a is expressed in Sf9 cells (Kiely et al.,
2007). This provides confidence that an insect cell assay system will be able to
provide a better environment for assaying moth receptors and functional
characterisation assays for moth ORs using this system has shown to confer high
sensitivity to the ORs for their respective ligands. Four female biased B. mori ORs
were expressed and tested with a panel of odorants of importance to the moth in the
Sf9 system. Two of these, BmOR45 and BmOR47 recognised the compounds 2-
phenyl ethanol and benzoic acid respectively to concentrations as low as 10 -14 M,
implicating the high sensitivity of the system, while BmOR30 did not respond to any
of the tested compounds and BmOR19 responded to linalool with an EC50 of 4.69 x
10 -9 M (Anderson et al., 2009). E. postvittana OR3 has also been de-orphaned using
the Sf9 system; it has been shown to recognise the oviposition deterrent of the moth,
the terpene citral to concentrations as low as 10 -15 M (Jordan et al., 2009). Unlike the
X. laevis oocyte and HEK293 cell assay systems mentioned above, the Sf9 cell assay
system does not require the co-expression of exogenous factors like OR83b and Gα15
protein. This is likely due to the presence of an endogenous homologue of Drosophila
OR83b in Sf9 cells as detected by RT-PCR (Smart et al., 2008).
Of the three ORs identified from E. postvittana, multiple sequence alignment and
phylogenetic analysis of EpOR1 with five other moth species placed EpOR1 in a
receptor clade that has 19 other OR members albeit with low homology (an overall
receptor amino acid identity of 10% in the clade, with 32–40% identity with
individual receptor sequences in the clade), as shown in Figure 1.8 (Jordan et al.,
2009). Some of these member ORs have been characterised for their expression
patterns in male and female moth antennae and many have been shown to be male

Functional characterisation of Epiphyas postvittana odorant receptor 1 39
biased, being highly expressed in male antennae with low or no expression in female
antennae. Functional odorant binding data is available for some of these ORs,
showing tuning of the ORs to the female sex pheromone components of the respective
species. The phylogenetic relatedness of EpOR1 to PRs from other moths make it a
candidate PR of E. postvittana. Quantitative real time polymerase chain reaction
(qRT-PCR) of EpOR1 was carried out on different male and female moth tissues and
the results showed that the expression was restricted to the antennae. Interestingly, no
sex-specific bias in expression levels of EpOR1 was observed, an indication that
EpOR1 was perhaps like HvOR6, BmOR9, PxOR3 and DiOR3, members of the clade
that do not have sex specific expression and may not respond to sex pheromone
components.
2.1.1 Aims
The aim of this research chapter is to deorphan EpOR1, to determine if EpOR1 is a
PR or an OR. If it is an OR then what compounds does it recognise and how does it
bind the different compounds that it recognises? Is EpOR1 broadly or narrowly tuned,
recognising a few or many compounds? How sensitive is EpOR1 to its ligands, is it
able to recognise low concentrations of odorants? To address these questions, EpOR1
is expressed and functionally characterised in the Sf9 cell assay system using calcium
imaging as it does not require the employment of exogenous factors for functional
expression of the receptor. EpOR1 is tested with the major pheromone component of
the sex pheromone blend of E. postvittana, as well as with a range of plant
semiochemicals of importance to the moth, as shown in behavioural studies and in
whole antennae EAG experiments (Suckling et al., 1996).
2.2 Materials and methods
2.2.1 Materials
Stock odorants were made at a concentration of 10 -2 M in DMSO and stored at -20°C
until required. The odorants used are as follows: -pinene (99%, racemic, BDH),
citral (95%, Sigma, mixture of neral (36%) and geranial (64%)), geraniol (97%,

Functional characterisation of Epiphyas postvittana odorant receptor 1 40
BDH), geranial (96.1%, synthesised by the method of Dess and Martin (1983),
geranyl acetate (98%, Sigma), nerol (97%, Sigma), linalool (97%, racemic, Sigma),
(+)-limonene (97%, Sigma), 1,8 cineole (90%, Fluka, Ronkonkoma, NY), myrcene
(Sigma), -terpineol (98%, Sigma), -farnesene (Sigma), caryophyllene (Koch-light
labs, Cambridge, MA), citronellol (95%, racemic, Sigma), humulene (ABD), octanol
(99.5%, Fluka), nonanol (98%, Fluka), hexanol (98%, Fluka), hexanal (98%, Sigma),
hexyl acetate (99%, Sigma), methyl salicylate (99%, Sigma), eugenol (BDH), ethyl
butyrate (Hopkin and Williams, Chadwell Health, Essex, UK), ethyl hexanoate (99%,
Sigma), pentyl acetate (99%, Sigma), dodecyl acetate (97%, Sigma), squalene (99%,
Sigma), pentanol (99.8%, Fluka), octanol (99.5%, Fluka), ethyl octanoate (99%,
Sigma), propyl propanoate (99.7%, Fluka), octyl acetate (99%, Sigma), propyl acetate
(99.7%, Fluka) and (E)-11-tetradecenyl acetate (97%, Sigma). Nerol, pentyl acetate,
octanol, geranyl acetate and methyl salicylate were stored at RT, while all remaining
compounds were stored at 4°C.
The assay saline buffer (pH 7.2) contained 21 mM KCl, 12 mM NaCl, 18 mM MgCl2,
3 mM CaCl2.H2O, 170 mM d-glucose, 1 mM probenecid (Sigma–Aldrich) and 10
mM PIPES-dipotassium salt. The assay saline buffer was sterilised by filtration
through a 0.22µM SteriCup–GP filter unit (Millipore) and stored at room temperature.
2.2.2 Insect cell culture
S. frugiperda Sf9 cells (Invitrogen) were maintained in shaker cultures in 50 mL
flasks at 28°C in the dark. The cells were grown to a density of 1 x 10 7 cells/mL in Sf
900 II serum free media (Invitrogen) after which they were seeded in new flasks at 1 x
10 5 cells/mL.
2.2.3 Transfection of cells
Viable cells were seeded at a density of 1 x 10 6 cells/mL in 12 well tissue culture
Nunclone plates with 400 µL Sf 900 II media. 0.5 µg pIB-EpOR1 (N-myc-tagged
EpOR1 in pIB-V5/His vector, constructed by Melissa Jordan) or the empty vector
control pIB-V5/His was incubated with 12 µL of the transfection reagent escort IV
(Sigma) and 100 µL of Sf 900 II media for each well at room temperature for 15

Functional characterisation of Epiphyas postvittana odorant receptor 1 41
minutes. This mixture was added to the well with the Sf9 cells and incubated at 27°C
in the dark for 7 hours. The cells were then washed and topped up with fresh media
and left to grow for a further 48 hours.
2.2.4 Detection of mRNA encoding pIB-EpOR1 by RT-PCR
A single well of each of the transfected cells (with pIB-V5/His or pIB-EpOR1) was
harvested 48 hours post transfection by removing the media and washing the cells
twice with 2 mL phosphate buffered saline (PBS). The cells were resuspended in 1
mL PBS, and pelleted by centrifugation at 5000g. The cells were stored at -80°C till
required. Total RNA was extracted using TRIzol reagent (Invitrogen) following
manufacturer‟s protocol, with the RNA resuspended in 10 µL diethylpyrocarbonate-
treated (DEPC-treated) water. Five microlitres of the RNA was used for synthesizing
cDNA using iScript cDNA synthesis kit (Bio-Rad) following the manufacturer‟s
protocol. A one in ten dilution of the cDNA was made and 2 µL of this was used in a
PCR reaction together with 1x PCR buffer including 1.5 mM magnesium
(Invitrogen), 0.2 mM of each dNTP (Invitrogen), 2 units of Taq DNA polymerase
(Invitrogen) and 0.5 µM of each primer. The final volume was made up to 20 µL with
sterile water. The forward and reverse primers used were EpOR1 gene specific
primers, EpOR1 forward– 5‟ATGGATGTATTCAATTTAAAATACATGCGA3‟ and
EpOR1 reverse– 5‟TCACTGATTTGCAAATGTTCTCAGCATCAG3‟, which
amplify the full length coding region of the EpOR1 cDNA (1248bp). The PCR
cycling conditions were as follows: initial denaturation at 94°C for two minutes,
followed by 35 cycles of 94°C for 20 seconds, 55°C for 30 seconds and 72°C for one
minute, and a final extension at 72°C for ten minutes. PCR products were analysed on
1% agarose gel stained with 1x SYBR safe DNA gel stain (Invitrogen) and images
captured on an ImageQuant 300 system (GE Healthcare Life Sciences).
2.2.5 Membrane Fraction Isolation and Western Blot
The method of isolation of membrane fractions was adapted from Bordier (1980). To
pre-condense Triton X-114, 5 mL of Triton X-114 was mixed with 250 mL PBS and
cooled to 0°C. The mixture was then heated at 37°C until the phases separated. The
top aqueous phase was discarded and 250 mL of PBS was added to the bottom phase

Functional characterisation of Epiphyas postvittana odorant receptor 1 42
and left on ice for 30 minutes. The mixture was heated at 37°C until phase separation
occurred and the aqueous phase was removed completely. The remaining lower phase
after removal of the aqueous phase was the pre-condensed Triton X-114 and this was
diluted to 2% (in PBS) for use in membrane extraction.
For extraction of the membrane proteins, the transfected Sf9 cells were washed twice
with ice cold PBS. The cells were then dislodged in 400 µL PBS, spun down at 3000g
for three minutes and the pellet resuspended in 200 µL Lysis buffer (2% pre-
condensed Triton X-114 made up in PBS). The cells were sonicated (2x 30 second
passes), incubated on ice for at least one hour and then spun at 13000g at 4°C for ten
minutes to remove insoluble debris. The supernatant was then transferred to a new
tube, incubated at 37°C for ten minutes, followed by centrifugation at 13000g at 25ºC
for ten minutes to separate phases. The aqueous phase was discarded and the bottom
layer was mixed with 1 mL of fresh cold PBS and incubated at 0°C for ten minutes.
The mixture was heated at 37°C for ten minutes to separate phases and centrifuged at
13000g at 25ºC for ten minutes. The aqueous phase was again discarded and the
washing steps repeated twice. The protein was resuspended in 25 µL of the following
mix for running on a western blot: 6.25 µL of NuPage LDS Buffer, 2.5 µL of NuPage
sample reducing agent, 16.25 µL of distil water and 6 M urea. It was then heated at
37°C for 20 minutes and spun at 13000g for 30 seconds to remove any particulates.
Ten microlitres was loaded on a NuPage 4–12% Bis-Tris gel (Invitrogen) and run at
200 volts for 35 minutes. The iBlot Dry Blotting System (Invitrogen) was used to
transfer protein from the gel to a nitrocellulose transfer membrane following
manufacturer‟s instructions. 20 mL of 1x milk powder in 1x PBS with 0.05% Tween
20 was used for blocking the membrane for two hours. The blocking buffer was
washed off with 1x PBS 0.05% Tween 20 and the membrane incubated with a one in
1000 dilution in 1x PBS with 0.05% Tween 20 with the primary antibody, anti-myc
mouse antibody (Roche) for one and half hours. The antibody solution was washed
off and the membrane washed three times with 1x PBS containing 0.05% Tween 20.
The membrane was then incubated with secondary antibody, goat-anti mouse IgGiAP
(Stressgen, diluted one in 2000) for one hour. The secondary antibody solution was
washed off and 1-Step NBT-BCIP (Nitro blue tetrazolium chloride 5-Bromo-4-
chloro-3-indolyl phosphate, toluidine salt, Pierce) substrate was added to visualise
bands on the membrane.

Functional characterisation of Epiphyas postvittana odorant receptor 1 43
2.2.6 EpOR1 functional assay
Functional assay of pIB-EpOR1 transfected Sf9 cells was carried out following the
method of Kiely et al. (2007). Briefly, 48 hours after transfection with EpOR1, the
cells were washed with assay buffer. This was done by tilting the plate, removing the
media with a pipette and adding 500 µL of assay buffer containing 2 μM Fluo-4
acetoxymethyl ester (Invitrogen) and 0.01% pluronic acid F–127 (Sigma-Aldrich) to
each well. The plate was incubated in the dark at 28°C for 20 minutes after which it
was washed with assay buffer to remove traces of any remaining dye. 400 µL of fresh
assay buffer was added to the wells and a second incubation at 28°C was done for 20
minutes. Calcium imaging of the Fluo-4 loaded cells was carried out on a Leitz
Fluovert FS inverted fluorescence microscope fitted with a MicroMax CCD camera
system (model RTE/CCD-1300-Y/HS; Princeton Instruments). An I3 filter set
(excitation filter 450-490 nm, dichroic mirror 510 nm, long pass emission filter 520
nm) was used to image the Fluo-4 labelled cells. MetaFluor v5.0 (Universal Imaging
Corporation) was used to operate the camera and control the image acquisition.
Transfected cells were tested with each of the odorants listed in section 2.2.1.
Odorants were made up to 0.1 M in DMSO and then further diluted in assay buffer.
Dose response data were collected for compounds that elicited consistent responses at
concentrations below 10 -5 M. Three wells on each 12 well Nunclone plate were tested
with the same concentration, and all the plates tested with a particular compound on a
given day were transfected using the same stock transfection mix.
To carry out calcium imaging of the cells, a field of view with evenly spread healthy
cells was selected in each well with the 10x objective lens. Six initial images were
taken at ten second intervals, then 50 µL of the assay buffer was added (to control for
cells that auto-fluoresce upon the addition of a solvent) and another six images
acquired. 50 µL of the test compound was then added, six further images were
acquired and finally 50 µL of the ionophore, ionomycin (Sigma) was added to a final
concentration of 10 µM to estimate the maximum possible fluorescence of the cells.

Functional characterisation of Epiphyas postvittana odorant receptor 1 44
2.2.7 Data analysis
The data collected from the calcium imaging was analysed using MetaFluor Analyst
v5.1 (Universal Imaging Corporation). Circles were drawn with the region of interest
(ROI) tool around the perimeter of the cells in the field of view and also three
background regions free of cells. The fluorescence data for the selected ROIs over the
image acquisition period was obtained with the „calculate plot‟ function of the
software. The data from the three background ROIs was averaged and the ROI data
from the cells was subtracted from this background, to minimise differences in cell
image backgrounds.
The change in fluorescence (∆F) of the cells that elicit a response upon odorant
stimulation was calculated as follows:
Where,
FBlank is the average fluorescence intensity of the cell, from the first image to the
twelfth image before addition of the ligand;
FSubstrate is the highest fluorescence intensity of the cell after the addition of the ligand,
from the thirteenth to the eighteenth image, and
FMax is the maximum fluorescence of the cell achieved after the addition of the
ionophore, from image nineteen to twenty-four.
The average ∆F of all the cells responding to a particular ligand concentration was
take as the mean ∆F for that concentration (with a minimum threshold of at least 3
responding cells) when plotting the dose response curve using OriginPro v7.5
(OriginLab) software. Cells that respond to stimulation with assay buffer only were
discarded from the analysis. EC50 (concentration at which 50% of the maximal effect
or activation of the ligand is observed) and Hill‟s slope (slope of the dose response
curve that gives the level of cooperativity of the ligands) (Motulsky and
Christopoulos, 2003) were calculated using Graphpad Prism software. A compound
was considered unable to activate EpOR1 if no response was observed in cells upon
stimulation with the odorant in triplicates at concentration of 10 -5 M.

Functional characterisation of Epiphyas postvittana odorant receptor 1 45
2.3 Results
To determine if the transfection of pIB-EpOR1 into Sf9 cells had been successful,
reverse transcription polymerase chain reaction (RT-PCR) was performed on mRNA
extracted from the transfected cells using primers specific for the full length coding
region of the EpOR1 gene (Figure 2.1A). As a control, cells transfected with the
pIB/V5 empty vector were also tested (Figure 2.1B). The empty vector transfected
cells did not show the presence of EpOR1 transcripts, while the EpOR1-transfected
cells showed the presence of PCR products of the expected size, 1245bp.
Figure 2.1: Reverse transcription PCR analysis of transfected pIB-EpOR1 Sf9 cells.
A. RT-PCR with myc-EpOR1 specific primers of pIB-EpOR1 transfected Sf9 cells 48
hours post transfection. B. RT-PCR of Sf9 cells transfected with the pIB/V5, an
empty vector control, with myc-EpOR1 specific primers.
To confirm the expression of myc-EpOR1 protein in transfected Sf9 cells, a western
blot of the membrane fraction from these cells was probed with an anti-myc antibody.
Three bands of approximately 25 kDa, 50 kDa and 60 kDa were observed on the
western blot (Figure 2.2). The band „b‟ observed at approximately 50 kDa
corresponds to the theoretical molecular weight of myc-tagged EpOR1. The higher
band „a‟ could be aggregation of the protein that did not separate out on the gel or a

Functional characterisation of Epiphyas postvittana odorant receptor 1 46
glycosylated form of the receptor protein while the lower band observed at „c‟ could
be degraded protein or background noise, as seen in the vector only lane 3.
Figure 2.2: Western blot of myc-tagged EpOR1 in Sf9 cells transfected with pIB-
EpOR1. Lane 1 is the magic marker western ladder (Invitrogen), lane 2 is the pIB-
EpOR1 protein membrane fraction and lane 3 is the pIB-V5/His protein membrane
fraction from cells transfected with vector only. Three bands of 60 (a), 50 (b) and 25
(c) kDa were observed in lane 2, the 50 kDa band corresponding to the molecular
weight of myc-EpOR1.
2.3.1 EpOR1 functional characterisation in Sf9 cells
In a typical calcium assay experiment, no change in fluorescence intensity of the Sf9
cells is observed upon addition of the control saline solution. An increase in the
intracellular calcium levels, which is measured as an increase in fluorescence of the
pIB-EpOR1 expressing cells is observed upon the addition of the ligand, and the
maximum fluorescence intensity of the cells is achieved upon addition of the
ionophore, ionomycin as depicted in Figure 2.3. Sf9 cells transfected with the empty

Functional characterisation of Epiphyas postvittana odorant receptor 1 47
pIB-V5/His vector were also tested in a dose-dependent manner with the methyl
salicylate and the data obtained is given in Appendix A.
Figure 2.3: The change in response of a single Sf9 cell transfected with pIB-EpOR1
over the timecourse of a calcium assay experiment. Arrow on the images indicates the
change in fluorescence intensity of the responding cell plotted on the graph over the
course of the assay: six initial images are acquired followed by the addition of the
control assay saline buffer represented in frames 6–12, then 10 -5 M citral is added and
six more images are acquired (frames 12–18). Frames 18–24 are acquired after the
addition of the ionophore, ionomycin.
Of the 33 compounds tested (Table 2.1), ten putative ligands were identified for
EpOR1, based on the ability of the compounds to elicit a calcium response at a
concentration of 10 -5 M in Sf9 cells transfected with pIB-EpOR1. These putative
ligands belong to a wide range of chemical groups ranging from monoterpenes
through to alcohols, esters and benzoates. The average normalised change in
fluorescence of the cells upon interaction with each ligand at 10 -5 M was calculated
and is given in Table 2.1.

Functional characterisation of Epiphyas postvittana odorant receptor 1 48
Table 2.1: Change in fluorescence at 10 -5 M of the compounds tested with EpOR1 in
the Sf9 cell assay. Each odorant was tested in triplicate from a single transfection mix.
Compound
Group
Compound Name ∆F a N c
Monoterpene Limonene NR b 0
Myrcene NR 0
-pinene NR 0
Linalool NR 0
Citronellol NR 0
-terpineol NR 0
1,8 cineole 0.43 ± 0.25 6
Nerol 0.41 ± 0.14 8
Geraniol 0.31 ± 0.03 8
Citral 0.32 ± 0.02 7
Geranial 0.36 ± 0.02 6
Geranyl acetate 0.32 ± 0.02 6
Sesquiterpene -farnesene 0.41 ± 0.12 7
Caryophyllene NR 0
Humulene NR 0
Triterpene Squalene NR 0
Alcohol Hexanol NR 0
Nonanol NR 0
Pentanol NR 0
Octanol 0.25 ± 0.21 6
Aldehyde Hexanal NR 0
Ester Ethyl butyrate NR 0
Ethyl octanoate NR 0
Ethyl hexanoate NR 0
Propyl propanoate NR 0
Pentyl acetate 0.37 ± 0.31 8
Hexyl acetate NR 0
Dodecyl acetate NR 0
Octyl acetate NR 0
Propyl acetate NR 0
(E)-11-tetradecenyl acetate NR 0
Benzoate Eugenol NR 0
Methyl salicylate 0.31 ± 0.03 6
a. Average change in fluorescence ± SE of at least three cells.
b. NR = no response as determined by no change in fluorescence of the cells
upon stimulation with the odorant in triplicates at 10 -5 M.
c. N = number of responding cells. A minimum of 300 cells were looked at to
confirm that a compound did not elicit a calcium response in the cells.

Functional characterisation of Epiphyas postvittana odorant receptor 1 49
To examine the sensitivity of EpOR1 for the ten responding ligands, calcium assays
were conducted over a range of ligand concentrations to enable the construction of
dose response curves. Ligands were tested at ten-fold decreasing concentrations
starting at 10 -5 M. Only five of the ten compounds (methyl salicylate, geranyl acetate,
citral, geraniol and geranial) elicited a calcium response at concentrations lower than
10 -5 M and the compounds differed in their levels of sensitivity as shown in Figure
2.4.
A relative measure of the difference between the binding affinities of EpOR1 to the
different ligands can be represented by comparing estimated EC50 values, the
concentration at which 50% of the maximal effect or activation of the ligand is
observed (Motulsky and Christopoulos, 2003). The level of cooperativity of the
ligands is given by the slope of the dose response curve and is calculated as the Hill
slope. The lowest concentration of ligand to which a response of the cells was
observed varied between the five best ligands and is given in Table 2.2, together with
the EC50 and Hill slopes from the dose response curves.

Functional characterisation of Epiphyas postvittana odorant receptor 1 50
A
C D
E
Figure 2.4: Dose response curves of the change in fluorescence exhibited by Sf9 cells
transfected with pIB-EpOR1 to (A) methyl salicylate, (B) geranyl acetate, (C) citral
(D) geraniol and (E) and geranial. The change in fluorescence of the pIB-EpOR1
transfected cells elicited by a range of ligand concentrations was calculated and the
data represented by a sigmoidal curve for each ligand. Error bars represent the
standard error of six to nine responding cells.
B

Functional characterisation of Epiphyas postvittana odorant receptor 1 51
Table 2.2: EC50 (± standard error) and Hill slope estimates of the dose response
curves of five best ligands for EpOR1. The recognition threshold is the lowest
concentration to which EpOR1 transfected Sf9 cells elicited a measurable response to
the test compound.
Compound EC50 (M) Hill slope Recognition threshold (M)
Methyl salicylate
Geraniol
Citral
Geranial
Geranyl acetate
2.4 Discussion
(1.8 ± 0.95) x 10 -12
(5.8 ± 0.35) x 10 -11
(1.3 ± 2.5) x 10 -9
(5.3 ± 2.5) x 10 -8
(2.8 ± 0.4) x 10 -8
0.35
3.2
0.33
0.61
0.83
10 -15
10 -12
10 -10
10 -11
10 -10
Using an insect cell assay system, E. postvittana OR1 was functionally characterised
to be a receptor for general plant odorants, including citral, geraniol, geranial, methyl
salicylate, geranyl acetate, pentyl acetate, octanol, α-farnesene, nerol and 1,4-cineole.
The presence of EpOR1 at the mRNA and protein levels in transfected Sf9 cells
(detected by RT-PCR and western blot respectively) suggests that EpOR1 is being
transcribed and translated in the Sf9 cells giving confidence that a functional assay is
possible. Western blot of membrane fraction extracted from Sf9 cells transfected with
pIB-EpOR1 showed three bands, one at approximately 25 kDa, the second at 50 kDa
and the third at 60 kDa. The 50 kDa band corresponds closely to the theoretical
molecular weight of myc-tagged EpOR1 at 49.6 kDa, while the higher 60 kDa band
could be a glycosylated form of the protein, as has been postulated by Henningsen et
al. (2002) for multiple bands on immunoblots of human histamine H2 receptor. The
lowest band could be degraded protein or nonspecific binding as the pIB-V5/His
transfected Sf9 cell membrane protein fraction (lane 3) also shows non-specific
binding around 25 kDa.
When the pIB-EpOR1 transfected Sf9 cells were stimulated with 10 -5 M citral, as
shown in Figure 2.3, not all the cells showed an increase in fluorescence. This is due

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

Functional characterisation of Epiphyas postvittana odorant receptor 1 54
odorant binding of EpOR1 can be seen in the Hill slopes of the dose response curves.
The Hill slope for geraniol is 3.2, suggesting a steep increase in binding with
increasing concentrations of the odorant, while the Hill slopes for the other
compounds are all below 1, suggesting shallower curves with a lower level of
cooperativity of the ligand into the receptor binding pocket.
EpOR1 is broadly tuned, recognising a range of compounds with different chemical
properties. Ligand responses at a concentration of 10 -5 M show the broad response
repertoire of EpOR1 (Table 2.1), from a seven carbon ester to a 15 carbon
sesquiterpene; from cyclic to acyclic compounds. This is comparable with results
obtained by Jordan et al. (2009) for EpOR3, which binds fifteen different compounds,
from a six carbon ester to a 15-carbon sesquiterpene. The broad tuning of EpOR1 at
10 -5 M is reduced to only five compounds when EpOR1 expressing Sf9 cells were
challenged with decreasing concentrations of the ten compounds listed in Table 2.1 in
a dose-dependent manner. These compounds, shown in Table 2.2 are 8-12 carbon
aromatics, with the smaller compounds (methyl salicylate, has eight carbons as
compared with geranyl acetate that has 12 carbons) eliciting higher sensitivity.
Perhaps the chemistry of the compound affects the binding specificity of the ORs, as
the results obtained by Hallem and Carlson, (2006) shows the preference for small
aromatic ring compounds by Drosophila OR49a. The activation threshold of EpOR3
by citral from Jordan et al. (2009) is 10 -15 M. This concentration is 10 5 -fold lower than
the threshold of EpOR1 for citral. The recognition of the same compound by the two
E. postvittana ORs suggests that the moth employs these two receptors in a
combinatorial manner that may help to expand the dynamic range of its olfactory
system. This combinatorial approach has also been observed in Drosophila, where a
range of ORs are able to detect the same odorants with different sensitivities, for
example, the compounds isobutyl acetate is detected by seven different ORs, and 2-
pentanone is detected by ten different ORs (Hallem and Carlson, 2006). This broad
tuning of ORs may be a mechanism for the insect to be able to detect more
compounds in various combinations, for example, to find suitable oviposition sites, so
as to lay eggs in close proximity of food source for the larvae, away from infested
plants, with the limited number of ORs that they have [from the genome sequence of
B. mori 68 ORs have been annotated so far, (Wanner et al., 2007; Tanaka et al.,
2009)]. This broad tuning of EpOR1 to its ligands as determined in the Sf9 cell assay

Functional characterisation of Epiphyas postvittana odorant receptor 1 55
might be very different to how the moth perceives these same ligands in the
environment and does not reflect that the moth will behave in a similar manner in the
environment. The combination of the hundreds of odorants present in the environment
together with the combinatorial mechanism of OR functioning might give varying
results to what is seen here.
EpOR1 also recognises odorants that are relevant to the biology of E. postvittana.
Geraniol for example, is a ten carbon monoterpernoid alcohol that has a rose-like
odour (Gochnauer et al., 1979). Geraniol is a deterrent that affects the mean number
of eggs laid by female E. postvittana and the site of oviposition (Suckling et al.,
1996). Citral is a ten carbon monoterpene which is a mixture of the cis and trans
isomers. Citral has previously been shown to act as an oviposition deterrent in E.
postvittana, that reduces the number of females laying eggs and also the number of
eggs laid per female (Suckling et al., 1996). Methyl salicylate is an eight carbon
organic ester that has a sweet woody odour. Plants produce this compound when they
are under herbivore attack (James and Price, 2004). Its release results in the
recruitment of beneficial insects that will kill the herbivorous insects. It is also used as
an alarm pheromone by plants that are under pathogenic attack to warn other plants of
the danger. The ability of EpOR1 to recognise this compound might be an adaptation
of E. postvittana towards screening out plants that are already infested with other
insects, thus avoiding these plants when looking for suitable oviposition sites and
choosing healthy non-infested plants where the competition for resources is less.
Geranyl acetate is a 12 carbon monoterpene and has a pleasant floral or fruity aroma
(Gildemeister and Hoffman, 1966). This odorant has been shown to enhance the flight
of grape berry moth towards its sex pheromone (Roelofs et al., 1971); hence it might
act as an attractant in E. postvittana also, directing the moth towards either food
sources or when combined with the sex pheromone, enhances the attraction of male
moths towards the female moths.
The same odorant receptor has similar binding specificities for attractants and
deterrents. One of the ligands for EpOR1, citral, having an EC50 of 1.3 x 10 -9 M, has
previously been shown to act as an oviposition deterrent for E. postvittana in
behavioural experiments (Suckling et al., 1996). At the same time, EpOR1 can bind
geranyl acetate (with an EC50 of 2.8 x 10 -8 M), which may act as an attractant for E.

Functional characterisation of Epiphyas postvittana odorant receptor 1 56
postvittana as discussed in the previous paragraph. There is only a ten-fold difference
in the binding affinity of these two compounds to the same OR that may elicit
opposite behaviour in the moth. Moreover, EpOR1 has the same recognition threshold
for both these compounds, recognising the compounds to a low concentration of 10 -10
M.
Functional characterisation of EpOR1 suggests that this is a general odorant receptor
tuned to recognising important plant volatiles. The level of tuning differs for each
odorant, with the sensitivity for methyl salicylate being the highest. Perhaps EpOR1 is
the main receptor that E. postvittana uses for the detection of methyl salicylate,
thereby identifying plants that are under attack. It will be interesting to see if any of
the other ORs in E. postvittana recognise methyl salicylate thus using a combinatorial
mechanism of binding for this ligand. The high affinity of insect ORs for plant
volatiles makes them potential targets for insect repellent development. Finally, the
identification and decoding of more E. postvittana ORs will enable the decoding of an
odour–ligand map for E. postvittana and aid in dealing with this important
agricultural pest.

3.1 Introduction
3
The roles of Epiphyas
postvittana GOBP2 in
odour detection by
EpOR1
The abundance of odorant binding proteins in the sensillum lymph poses numerous
questions about their function. Although OBPs were discovered in insects nearly two
decades before the odorant receptors, there continues to be much debate over their
roles(s). Several roles for OBPs have been postulated based on experimental data
obtained from studies of PBPs. These proteins form a family with 32–92% sequence
identity in moths (Abraham et al., 2005). They are present at high concentrations in
the range of 10 µM in the sensillum lymph (Klein, 1987) and have a role in
pheromone binding (Vogt and Riddiford, 1981; Vogt et al., 1989; Maibeche-Coisne et
al., 1997; Newcomb et al., 2002). OBPs are thought to form complexes with ligands
and have been suggested to: aid the solubilisation and transport of ligands; protect
ligands from enzymatic degradation in the sensillum lymph; enable interaction of
ligands with dendritic bound ORs, and facilitate deactivation of the ligand (see section
1.5.1 for a detailed overview on the roles of OBPs).

The roles of Epiphyas postvittana GOBP2 in odour detection 58
Different methods have been used to examine the roles of PBPs both in vivo and in
vitro. Electrophysiological recordings of single antennal branches in A. polyphemus
upon stimulation with pheromone have shown that ApolPBP decreases the
concentration of pheromone required to elicit ORN response by 100-fold, hence
indicating a role of ApolPBP as a solubiliser of the pheromone (van den Berg and
Ziegelberger, 1991). In in vitro characterisation assays using calcium imaging of
HEK293 cells expressing BmOR1 and HvOR13, organic solvents were successfully
replaced by the respective species‟ PBP as the solubiliser of the pheromone (Große-
Wilde et al., 2006; Große-Wilde et al., 2007). A. polyphemus PBP has also been
shown by in vitro binding assays to act as a protector of the pheromone, as measured
by reduced activity of PDE on radio-labelled pheromone in the presence of PBP (Vogt
et al., 1985). Several assays have provided evidence for interaction of the OBP/ligand
complex with the dendritic bound ORs. The ab3A neuron of Drosophila has been
used for expressing BmOR1 and sensitivity of the OR to its ligand bombykol was
enhanced upon co-expression of BmPBP in the empty neuron (Syed et al., 2006).
Similarly, when BmOR1 was expressed in HEK293 cells, replacement of the organic
solubiliser DMSO with BmPBP selectively showed activity to BmPBP-bombykol
complex and the DMSO solubilised activity of bombykal was lost, as measured by
calcium imaging of the BmOR1 expressing HEK293 cells (Große-Wilde et al., 2006).
In vitro fluorescence measurements of endogenous tryptophan residues of BmPBP in
complex with bombykol at physiological pH of 7.0 and dendritic pH of 4.7 showed a
decrease in fluorescence indicating a dissociation of the OBP/ligand complex at low
pH (Leal et al., 2005). A similar study has been done in A. polyphemus to show
conformational changes of ApolPBP at different pH values (Mohanty et al., 2003;
Mohanty et al., 2004).
Other characterisation assays have also been used to deorphan insect OBPs. These
include the cold binding assay, a two-phase binding assay and a volatiles odorant
binding assay (Briand et al., 2001; Leal et al., 2005; Zhou et al., 2009). The cold
binding assay allows for testing binding of ligands to OBPs at different test conditions
such as pH and temperature and is able to separate free ligand from protein bound
ligand using a centrifugal filter device (Leal et al., 2005; Zhou et al., 2009). In this
assay, the test odorants are added to the protein solution and the mixture left to
incubate. The mixture is then centrifuged to remove unbound odorants in the filtrate

The roles of Epiphyas postvittana GOBP2 in odour detection 59
while the protein bound ligands are maintained in the retentate. The ligand is released
from the protein by altering the pH of the mixture and eluted in a layer of hexane,
which is then analysed for the presence of the ligands that were bound to the protein
by gas chromatography (GC). Recently, Zhou et al. (2009) proposed the two-phase
binding assay, due to the potential release of the ligand during the washing step in
cold binding assay. In this assay, the ligands are dissolved in a layer of hexane that
covers the protein solution, and the depletion of the odorants in the hexane layer is
measured as the uptake of odorants by the proteins in the bottom layer.
The identification of ligands for proteins, especially if the test odorants are
hydrophobic volatiles becomes difficult due to the tendency for such odorants to
adhere to surfaces such as test vial walls and if mixed directly to the solution, may
form micelles. The technique developed in the volatiles odorant binding assay
(VOBA) circumvents these problems by introducing the volatile slowly into the
protein solution via diffusion through a gas phase intermediate (Briand et al., 2001).
This technique was successfully developed and used by Briand et al. (2000) to
characterise an OBP isolated from rat nasal mucus. It has also been shown to facilitate
the uptake of a range of compounds by the honeybee Apis mellifera OBP (Briand et
al., 2001). The VOBA technique introduces the volatile slowly, molecule by molecule
so the distribution of odorants in the protein solution is uniform. An excess of the
odorant is used and the reaction is allowed to reach equilibrium so any adherence of
the odorant to the walls of the test vial becomes negligible.
In E. postvittana, three PBPs and one GOBP were identified from native protein gel
analysis and one PBP and another GOBP each identified from EST sequences
(Newcomb et al., 2002; Jordan et al., 2008). All the E. postvittana PBPs and one
GOBP have been shown on western blot and by qRT-PCR analysis to be expressed in
both male and female antennae, albeit with varying levels. Two of the PBPs were
shown to have higher expression in male than female antennae. Further sequence
analysis revealed them to be allelic variants of the same PBP, consisting of six amino
acid substitutions and differing in their electrophoretic properties, thus they were
named PBP1-fast and PBP1-slow. Both the PBP1 proteins bind the major sex
pheromone component of E. postvittana, (E)-11-tetradecenyl acetate in gel assays.
The third PBP, called PBP2 was shown to have higher expression in female than male

The roles of Epiphyas postvittana GOBP2 in odour detection 60
antennae hence postulated to have a female oriented role, perhaps in the binding of
some as yet unknown male specific pheromone. The fourth PBP, called PBP3 showed
higher expression in male than female antennae, and has been postulated to be
involved in binding of the pheromone component(s) of E. postvittana.
The two GOBPs are members of the GOBP1 and GOBP2 family of GOBPs in moths
and GOBP2 has a similar level of expression in male and female antennae (Newcomb
et al., 2002). No data on sensillum localisation of EpGOBPs is available yet, however,
the expression of A. polyphemus, B. mori and H. armigera GOBPs have been shown
to be restricted to sensilla basiconica, hence a role of GOBPs in binding plant volatiles
of importance to moths is assumed (Steinbrecht et al., 1995; Wang et al., 2003). Very
limited functional data is as yet available for any moth GOBP, with the only study
revealing that M. sexta GOBP2 binds the plant volatiles (Z)-3-hexen-1-ol, geraniol,
geranyl acetate and limonene (Feng and Prestwich, 1997) and a recent study revealed
B. mori GOBP2 binds bombykol and is able to discriminate it from the antagonist,
bombykal (Zhou et al., 2009). Both M. sexta and B. mori GOBP1 have so far failed to
bind any of the ligands they have been tested with (Vogt et al., 2002; Zhou et al.,
2009) leading to the conclusion that the proteins tested might be inactive allelic
variants.
3.1.1 Aim
The aim of this research chapter is to test hypotheses of possible roles for GOBPs.
GOBPs could be non-specific solubilising agents for a range of plant volatiles, or they
could be ligand specific, like that observed for moth PBPs. GOBPs could form a
complex with the ligand, and interaction of this complex confers increased sensitivity
and selectivity onto the OR, as shown for H. virescens and B. mori PBPs in
heterologous cell assay systems. With this knowledge of roles of PBPs in
heterologous systems, a similar approach of using a reconstituted heterologous assay
system can be used to test the hypothesised roles of GOBPs with E. postvittana
GOBP2 (EpGOBP2) as a model: can EpGOBP2 act as a solubilising agent for plant
volatiles? GOBP2 homologues in M. sexta and B. mori have been shown to bind plant
volatiles and sex pheromone components hence we assume EpGOBP2 to have a
similar ligand binding range. As part of this PhD research, E. postvittana OR1 was

The roles of Epiphyas postvittana GOBP2 in odour detection 61
characterised in an Sf9 cell assay system and shown to bind a range of plant volatiles.
Some of the plant volatiles identified as ligands of EpOR1 have also been shown to be
ligands for M. sexta GOBP2 (geraniol and geranyl acetate); hence leading us to
hypothesis that EpGOBP2 might share ligands with EpOR1. The odorants used for
testing EpGOBP2 hence is limited to the binding repertoire of EpOR1, as determined
in chapter two. The Sf9 cell assay system heterologously expressing EpOR1 forms a
good starting point for testing the role(s) of EpGOBP2.
3.2 Materials and methods
3.2.1 Recombinant Expression of EpGOBP2
A pET30a (Novagen) plasmid clone of EpGOBP2 (pET30a-EpGOBP2) was obtained
from Duncan Stanley at Plant & Food Research. This clone encodes a protein
containing an N-terminal His-tag and TEV protease recognition site upstream of the
EpGOBP2 sequence (411 bp, GenBank accession no. AF411460), which is missing
its N-terminal 20 amino acid signal peptide (Newcomb et al., 2002).
The pET30a-EpGOBP2 plasmid was transformed into Rosetta-Gami2 cells (Novagen)
and positive clones selected on kanamycin and chloramphenicol luria broth (LB)
plates. pET30a-EpGOBP2 was over-expressed in buffered terrific broth (TB) with
isopropyl-beta-D-1 thiogalactopyranoside (IPTG) induction, as stated in Hamiaux et
al. (2009). Refer to appendix A for recipe of TB. Briefly, a colony of the transformed
cells was used to inoculate a starter culture for protein expression consisting of 10 mL
LB media, 15 µg/mL kanamycin, 34 µg/mL chloramphenicol and 0.01% glucose.
This was incubated at 37°C overnight with shaking at 220 rpm after which 5 mL of
the culture was used to inoculate 500 mL of buffered TB with 15 µg/mL kanamycin
and 34 µg/mL chloramphenicol. The culture was left to grow at 37°C, 220 rpm until
an optical density (OD600) of 0.6 to 0.8 was attained. IPTG was then added to the
culture to a final concentration of 0.5 mM, and the culture let to grow overnight at
20°C shaking at 220 rpm. The cells were harvested by centrifugation at 13,000g at
4°C for 30 minutes, with the resulting pellet stored at -20°C until purification.

The roles of Epiphyas postvittana GOBP2 in odour detection 62
3.2.1.1 Purification of recombinant EpGOBP2
The cell pellet was resuspended in 20 mL HisTrap binding buffer (consisting of 20
mM Tris pH 8.0, 500 mM NaCl and 20 mM imidazole) containing EDTA free
protease inhibitors (Roche). This solution was emulsified at 15000 psi by passing the
samples through an emulsiflex-CS high-pressure homogeniser (Avestic Inc.) three
times to get maximum cell lysis. The sample was then centrifuged at 13,000g at 4°C
for 30 minutes, the resulting pellet discarded and the soluble fraction filtered through
a 0.22 µm filter (Millipore) to remove any remaining cell debris and kept on ice until
purification.
Purification of the His6-EpGOBP2 was carried out on a 5 mL HiTrap chelating HP
column (GE Healthcare) by affinity chromatography on ÄKTAprime fast protein
liquid chromatography (GE Healthcare). The protein solution was loaded onto the
column equilibrated in HisTrap binding buffer containing 20 mM imidazole. Bound
His6-EpGOBP2 was eluted from the column by a 20 to 250 mM imidazole linear
elution gradient. The fractions containing His6-EpGOBP2 were identified by running
on 4-12% Bis-Tris SDS PAGE gel (Invitrogen) and visualised using colloidal
commassie G-250 blue stain (Neuhoff et al., 1988). Fractions showing the presence of
His6-EpGOBP2 were pooled and dialysed overnight into ion exchange binding buffer
consisting of 20mM Tris HCl, pH 8 and 25 mM NaCl and loaded onto a Q sepharose
HP column (GE Healthcare). Bound protein was eluted from the column by a 25 to
500 mM NaCl linear elution gradient. The fractions containing His6-EpGOBP2 were
identified by SDS-PAGE analysis, pooled and then dialysed against 50 mM Tris-HCl
pH 8.0, 0.5 mM EDTA overnight. After dialysis, the His6-tag was cleaved off with
AcTEV Protease (Invitrogen) following manufacturer‟s instructions. After cleavage,
the protein sample was dialysed into HisTrap binding buffer for purification of the tag
free protein on the HiTrap chelating HP column as stated above, this time
collecting the protein in the flow through.
3.2.1.2 Delipidation of recombinant EpGOBP2
Recombinant OBPs have a tendency to bind exogenous ligands from the expression
cells hence these artefact ligands have to be removed from the proteins before any

The roles of Epiphyas postvittana GOBP2 in odour detection 63
functional analysis is carried out (Oldham et al., 2000; Hamiaux et al., 2009). The
tag-cleaved protein was delipidated according to Oldham et al. (2001). Briefly, the
protein was concentrated to 50 µL using a Vivaspin ultrafiltration concentrator
(capacity 500 µL, MWCO 5 kDa) at 10,000 rpm, 4°C (Vivaproducts, MA). 500 µL of
25 mM ammonium acetate pH 4.5 was then added to the retentate and centrifuged
again to 50 µL. This was repeated a second time and then 350 µL of 25 mM
ammonium acetate pH 4.5 was added again to the retentate and placed on ice. 500 µL
of the Lipidex 1000 methanol suspension (Perkin Elmer) was centrifuged in a MC-
Ultrafree centrifuge filter cup (capacity 500 µL, pore size 0.22 µm, Millipore,
Bedford, MA) at 10,000 rpm for one minute to remove the liquid phase. The flow
through was discarded and another 500 µL of Lipidex was added to the filter cup,
centrifuged and the flow through discarded again. The Lipidex in the filter cup was
mixed with 500 µL of 25 mM ammonium acetate pH 4.5, centrifuged at 10,000 rpm,
for one minute and the flow through discarded. This was repeated again and then the
EpGOBP2 sample was mixed thoroughly with the Lipidex, and the resulting
suspension incubated in the filter cup at 37°C for one hour with gentle shaking. The
suspension was then centrifuged at 10,000 rpm, 4°C; the flow through collected and
kept on ice. The Lipidex was washed with 500 µL of 25 mM ammonium acetate pH
4.5 (to remove any remaining traces of EpGOBP2), centrifuged and flow through
collected again. Both the flow through samples were combined and concentrated
using the Vivaspin ultrafiltration concentrator to 50 µL. The concentrated, delipidated
EpGOBP2 was diluted in 2.5 mM ammonium acetate at neutral pH to a concentration
of 100 µM and dialysed against 50 mM Tris-HCl buffer pH 7.5 for conducting
binding assays.
3.2.2 Volatile odorant binding assay
The volatile odorant binding assay (VOBA) (Briand et al., 2000) was used for testing
the ligand preferences of EpGOBP2. Fifty microlitres of 5 µM protein in 50 mM Tris-
HCl, pH 7.5 or 50 µL of 50 mM Tris-HCl buffer pH 7.5 (control) were setup in
triplicates in 200 µL PCR tubes. The tubes were placed with lids open in Conway
microdiffusion dish (Gallenkamp). A drop of the absolute compound to be tested was
added to the dish and the system was sealed with a glass lid. The system was left to
equilibrate for approximately 12 hours at room temperature, as depicted in Figure 3.1.

The roles of Epiphyas postvittana GOBP2 in odour detection 64
Figure 3.1: Schematic representation of the VOBA setting to show the distribution of
the odorant at equilibrium in a sealed chamber.
The tubes were removed after the 12 hour incubation. To release the bound
compounds, 5 µL of 1 mg/mL proteinase K (Roche) was added to the tube content,
which was then covered with a layer of hexane (to capture odorants released from
digested protein). This was done for all the tubes, including the buffer only control. 5
µM of an internal standard, methyl tetradecanoate (14OMe) was added to all the
tubes. The closed tubes were then incubated at room temperature for two hours. The
hexane layer was removed from tubes into clean 2 mL glass vials and analyzed by GC
using a HP 6890 Series GC System (Hewlett Packard) equipped with an on-column
injector and FID detector (300°C). The analytical column used was a HP-INNOWax
Polyethylene Glycol (nominal length = 30.0 m, nominal diameter = 250.00 µm and
nominal film thickness = 0.25 µm; Agilent Technologies). The oven temperature
gradient was applied from 120°C to 260°C at 5°C.min -1 . The carrier gas was helium at
0.7mL.min -1 . Calibration was done by odorants diluted in chloroform.
The concentration of the compounds was determined by measuring surface of peaks
with respect to the calibration curve. The concentration of the bound ligand and the
binding affinity (dissociation constant, ) of the ligands with the protein were
calculated using the method of Lazar (2001) as follows:
Protein
Test ligand
Buffer

The roles of Epiphyas postvittana GOBP2 in odour detection 65
From the results obtained in the GC,
[L] = free ligand (total ligand present in buffer only)
[P] = protein concentration used for VOBA (5 µM)
[PL] = concentration of bound ligand (Total ligand present in protein sample – [L])
= dissociation constant (the smaller the , the tighter the binding)
3.2.3 Reconstituted cell assay
The reconstituted cell assay was carried out as described in Groβe–Wilde et al. (2006)
with a few modifications. Briefly, odorants were made up in the concentration range
of 10 -5 M to 10 -14 M as described in section 2.2.6 in either assay saline alone or assay
saline containing 10 -6 M EpGOBP2 solution. The Sf9 cell transfection, assay and data
analysis were performed as stated in sections 2.2.6 and 2.2.7.
3.3 Results
3.3.1 Purification of recombinant GOBP2
Recombinant EpGOBP2 was purified in order to carry out functional ligand binding
assays to deduce its ligands and also to test its role(s) in odorant binding of EpOR1 in
the Sf9 cell assay system. His6-EpGOBP2 eluted as a single peak from 60 to 75%
imidazole. The six fractions collected during this elution peak were run on 4-12%
SDS-PAGE gel (Figure 3.2). No EpGOBP2 was present in the flow through
indicating all the EpGOBP2 loaded onto the column was bound to it. However, not all
of the EpGOBP2 separated into the soluble phase as there is still a thick band in the
insoluble fraction at 22.6 kDa.

The roles of Epiphyas postvittana GOBP2 in odour detection 66
Figure 3.2: Purification of His6-EpGOBP2 by HiTrap chelating HP column. A
dominant EpGOBP2 band is observed at 22.6 kDa in lanes 4–9. Lane 1 is the
insoluble fraction containing the cell debris, lane 2 is the crude protein sample that
was loaded onto the column and lane 3 is the flow through.
His6-tagged EpGOBP2 was present in all six fractions over the elution peak, shown
by a dominant band at 22.6 kDa. However, other bands were present in the fractions
also, possibly E.coli proteins that interacted with the nickel column. To further purify
EpGOBP2, the six fractions from the HiTrap chelating column were combined and
dialysed into ion exchange binding buffer for purification on Q-sepharose HP column
(Amersham Biosciences). The protein was eluted using a gradient of 500 mM NaCl.
The seven elution peak fractions obtained were run on 4–12% a SDS-PAGE gel as
shown in Figure 3.3.

The roles of Epiphyas postvittana GOBP2 in odour detection 67
Figure 3.3: Purification of His6-EpGOBP2 from the pooled HiTrap fractions by Qsepharose
anion exchange chromatography. Lane 1 is the EpGOBP2 dialysed into ion
exchange binding buffer and lanes 2–8 are the elution peak fractions from the Qsepharose
HP column containing the His6-EpGOBP2.
The fractions obtained after the ion exchange were pooled and the His6-tag was
cleaved off by AcTEV digestion. After tag removal, the protein was recovered by
running the sample on HiTrap chelating HP column, this time collecting the protein
in the flow through (Figure 3.4I). The protein sample was finally put through a
delipidation procedure to remove any exogenous ligands that bound to the protein
from the E. coli cells (Figure 3.4II).

The roles of Epiphyas postvittana GOBP2 in odour detection 68
Figure 3.4: Purification of AcTEV cleaved EpGOBP2 by HiTrap chelating column
chromatography. (I) is the EpGOBP2 after treatment with AcTEV protease. Lane 1 is
the protein sample that was loaded onto the column, lane 2 is the protein bound to the
nickel column and lane 3 is the flow through from the Nickel column. Band A is the
His6-EpGOB2, band B is EpGOBP without the His6 tag and band C is the His6-tag.
(II) is the protein without the tag after going through the delipidation process.
3.3.2 Volatile Odorant Binding Assay
Ligands that bound to EpGOBP2 were identified using the volatile odorant binding
assay (Briand et al., 2000). The binding affinity of EpGOBP2 to a panel of ten
odorants that EpOR1 recognises was determined. EpGOBP2 was found to bind seven
out of the ten compounds that were tested in the micromolar range (Figure 3.5).

The roles of Epiphyas postvittana GOBP2 in odour detection 69
Figure 3.5: Overnight VOBA of EpGOBP2 with the ligands of EpOR1 indicating
that EpGOBP2 binds seven of these compounds in the micromolar range. The error
bars represent standard errors calculated from triplicate repeats. * = no detectable
binding.
Figure 3.6: Dissociation constants ( ) for ten odorants against EpGOBP2. are
the average from 3 replicates with error bars representing standard errors. * = no
detectable binding.

The roles of Epiphyas postvittana GOBP2 in odour detection 70
For the seven ligands identified in the scope of this study, octanol had the highest
binding capacity for EpGOBP2, with 99 µM of octanol binding to 5 µM of EpGOBP2
while only 9 µM of pentyl acetate bound to the same concentration of EpGOBP2
(Figure 3.5).
Ten compounds were identified as ligands for EpOR1 in Chapter 2, however, at lower
concentrations, EpOR1 was able to elicit a response to only five of these (geranyl
acetate, citral, methyl salicylate, geraniol and geranial) hence dose response curves
were able to be obtained for only these five compounds. From these five ligands of
EpOR1, geranyl acetate and methyl salicylate are able to bind to EpGOBP2, hence
both these compounds were used for testing the response of EpOR1 expressing Sf9
cells by reconstituting the Sf9 cell assay system with EpGOBP2.
3.3.3 Reconstituted EpOR1 receptor activation assays
In order to carry out reconstituted EpOR1 receptor activation assays in the Sf9 assay
system with EpGOBP2, the common ligands of both EpGOBP2 and EpOR1 for
which dose response curves have been determined in Chapter 2 are used. These
ligands are methyl salicylate and geranyl acetate. The dose response of EpOR1
expressed in the Sf9 cell assay to methyl salicylate and geranyl acetate was measured
under the following conditions: in the absence of both EpGOBP2 and DMSO (Figures
3.8A and 3.9A, no solubilising agent present hence the odorant does not elicit a
response in the EpOR1 expressing cells); in the presence of EpGOBP2 only (Figures
3.8C and 3.9C, a dose response of the EpOR1 expressing cells is observed to both
geranyl acetate and methyl salicylate indicating that the GOBP2 is able to solubilise
these two odorants); or DMSO only (Figures 3.8B and 3.9B, DMSO acts as a
solubilising agent for methyl salicylate and geranyl acetate as determined in Chapter
2); and in the presence of both DMSO and EpGOBP2 (Figures 3.8D and 3.9D, to
determine whether the changes in the EC50 values of the various dose response curves
are due to the presence of EpGOBP2 or DMSO). These sets of experiements showed
that EpGOBP2 can act as a solubilising agent for methyl salicylate and geranyl
acetate. To show that the dose response observed for both methyl salicylate and
geranyl acetate in the presence of EpGOBP2 is not due to a concentration effect of the

The roles of Epiphyas postvittana GOBP2 in odour detection 71
EpGOBP2, a no-odorant dose response control assay of the EpOR1 expressing Sf9
cells was carried out. This showed that in the absence of a ligand, EpGOBP2 does not
elicit a response in EpOR1 expressing Sf9 cells, as depicted in Figure 3.7. The
concentrations at which half the maximal responses (EC50) were obtained for the
different test concentrations is given in Table 3.1.
Figure 3.7: Dose response of EpOR1 expressing Sf9 cells to EpGOBP2 in the
concentration range of 10 -5 M to 10 -13 M. Error bars represent the standard error of six
to eight cells.

The roles of Epiphyas postvittana GOBP2 in odour detection 72
0.35
0.30
0.25
0.20
∆F
0.15
0.10
0.05
0.00
0.35
0.30
0.25
0.20
∆F
0.15
0.10
0.05
0.00
A
-12 -10 -8 -6 -4
[Geranyl Acetate] (Log M)
-12 -10 -8 -6 -4
[Geranyl Acetate] (Log M)
C
0.35
0.30
0.25
0.20
∆F
0.15
0.10
0.05
0.00
0.35
0.30
0.25
0.20
∆F
0.15
0.10
0.05
0.00
B
-12 -10 -8 -6 -4
[Geranyl Acetate] (Log M)
D
-12 -10 -8 -6 -4
[Geranyl Acetate] (Log M)
Figure 3.8: Dose response curves of EpOR1 to geranyl acetate in the concentration
range of 10 -5 M to 10 -12 M in the absence of both DMSO and EpGOBP2 (A), with
DMSO (B), or with EpGOBP2 (C), or with geranyl acetate solubilised in DMSO and
the cells incubated with EpGOBP2 (D). Error bars represent the standard error of six
to eight responding cells.

The roles of Epiphyas postvittana GOBP2 in odour detection 73
0.35
0.30
0.25
0.20
∆F
0.15
0.10
0.05
0.00
0.35
0.30
0.25
0.20
∆F
0.15
0.10
0.05
0.00
A
-16 -14 -12 -10 -8 -6 -4
[Methyl Salicylate] (Log M)
C
-16 -14 -12 -10 -8 -6 -4
[Methyl Salicylate] (Log M)
0.35
0.30
0.25
0.20
∆F
0.15
0.10
0.05
0.00
0.35
0.30
0.25
0.20
∆F
0.15
0.10
0.05
0.00
B
-16 -14 -12 -10 -8 -6 -4
[Methyl Salicylate] (Log M)
D
-16 -14 -12 -10 -8 -6 -4
[Methyl Salicylate] (Log M)
Figure 3.9: Dose response curves of EpOR1 to methyl salicylate in the concentration
range of 10 -5 M to 10 -16 M in the absence of both DMSO and EpGOBP2 (A), with
DMSO (B), or with EpGOBP2 (C) or with methyl salicylate solubilised in DMSO and
the cells incubated with EpGOBP2 (D). Error bars represent the standard error of six
to eight responding cells.

The roles of Epiphyas postvittana GOBP2 in odour detection 74
Table 3.1: A comparison of the EC50 values from dose response curves of EpOR1
obtained in Sf9 cell assays with DMSO and/or EpGOBP2 used as a solubilisation
agent for the ligands methyl salicylate and geranyl acetate.
Ligand components EC50[Methyl Salicylate] (M) EC50[Geranyl Acetate] (M)
Saline only NR a NR
DMSO only (1.8 ± 0.9) -12 (2.7 ± 0.4) -8
EpGOBP2 only (1.19 ± 0.82) -13 (1.9 ± 0.66) -10
DMSO + EpGOBP2 (1.33 ± 0.96) -13 (1.97 ± 2.5) -10
a. NR = no response
3.4 Discussion
Insects can recognise a range of chemicals, most of which are hydrophobic in nature.
The actual mechanism of odour recognition by insects is yet to be deduced; however
theoretical models suggest solubilisation and targeting of the hydrophobic odours to
the receptor neurons via soluble protein intermediates present in the sensillum lymph
(Kaissling, 2009). We can test the roles of these soluble proteins in an in vitro setting
by expressing the receptor in cell lines such as the Sf9 insect cell lines used in this
study, and looking at the effect on receptor activation upon addition of these proteins.
The ability of EpGOBP2 to solubilise plant volatiles of importance to E. postvittana
was tested in a VOBA setting. The ten compounds tested were chosen based on their
ability to activate EpOR1 expressed in the Sf9 cell assay system. EpGOBP2 was able
to solubilise seven of these compounds; however the binding repertoire of EpGOBP2
may be more extensive if the range of tested compounds was to be expanded. For the
scope of this study, only those compounds that elicited response to EpOR1 were
chosen for testing their binding to EpGOBP2.
The chemistries of these seven compounds differ considerably. Pentyl acetate is an
ester that has a straight chain structure, while geranyl acetate and nerol are linear
monoterpenes, eucalyptol is a cyclic monoterpene and α-farnesene is a sesquiterpene.
Methyl salicylate is a benzoic ring phenol and octanol is a linear alcohol (O'Neil,
2006). The amount of each of these ligands bound by EpGOBP2 differs according to
their chemistries, with EpGOBP2 having highest affinity for alcohol followed by the

The roles of Epiphyas postvittana GOBP2 in odour detection 75
phenol, then the terpenes and lastly the ester (Figure 3.5). However, the difference in
the degree of binding of these ligands is small, with only a ten-fold difference
between the highest octanol, and the lowest, pentyl acetate. Nevertheless, all these
compounds are plant volatiles and may play a role in E. postvittana olfaction (as
discussed in chapter 2).
The moderate binding constants of EpGOBP2 for the range of plant volatiles tested
reflect low affinity which supports the conclusion that GOBPs function to bind a
range of odorants. These moderate binding constants of EpGOBP2 for a range of
volatiles are indicative of a role in binding of a range of odorants by OBPs. Previous
studies of GOBPs in other lepidopterans have shown their ability to recognise plant
volatiles. M. sexta GOBP2 binds plant odorants such as (Z)-3-hexen-1-ol, geraniol,
geranyl acetate and limonene (Feng and Prestwich, 1997) with moderate affinities (10
to 40 µM of these odorants were required in displacement assays to displace 50 % of
the bound pheromone analog), comparable to those obtained for EpGOBP2 in this
study. In the honeybee Apis mellifera, the OBP called ASP2 binds its ligands, mostly
plant scents, with high binding affinities in the micromolar range (Briand et al., 2001).
This shows that perhaps the Hymenoptera and Lepidoptera OBPs differ in their
mechanisms of binding their ligands. Since EpGOBP2 binds a range of compounds
with different shapes and chemical structures, it can be deduced that the binding
pocket of EpGOBP2 must be spacious and flexible enough to harbor this varied range
of compounds. The range of compounds tested in this study is limited; hence there
might be other stronger binding ligand(s) that have not been tested yet. Increasing the
test compound range will perhaps give insights into an even wider range of ligands for
EpGOBP2, adding to the observation that while the PBP recognise species specific
pheromones, the GOBPs are broadly tuned to recognise a range of compounds that
includes (and might not be limited to) plant volatiles.
When EpGOBP2 was pre-incubated with geranyl acetate or methyl salicylate, and
added to Sf9 cells expressing EpOR1, a receptor activation response was observed
(Figures 3.8 and 3.9), indicating that EpGOBP2 was able to solubilise the odorants
enabling it to interact with the membrane bound EpOR1. This is the first documented
role of a lepidopteran GOBP in odorant binding in an in vitro OR characterisation
assay. Similar reconstitution assay studies have been done with lepidopteran PBPs,

The roles of Epiphyas postvittana GOBP2 in odour detection 76
namely BmPBP1 and HvPBP2 (Große-Wilde et al., 2006; Große-Wilde et al., 2007).
In these studies, the PBPs were shown to be able to replace the DMSO as a
solubilising agent for the sex pheromone components tested, and obtain functional
data for pheromone receptors when tested in a heterologous expression system.
Despite the different types of odorants GOBP2 binds when compared to these
narrowly tuned PBPs, the EpGOBP2 was able to replace DMSO in EpOR1
characterisation assays, acting as a solubilising agent for methyl salicylate and geranyl
acetate.
When DMSO was replaced by EpGOBP2 in the Sf9 cell assay, the EC50 value
decreased (Table 3.1), indicating that in the presence of EpGOBP2, EpOR1
expressing cells were able to bind more odorants at lower concentrations compared to
when DMSO was used. This was true for both methyl salicylate and geranyl acetate
(Table 3.1). The response was not a spontaneous response to EpGOBP2 as assays
carried out with EpGOBP2 alone did not show any response (Figure 3.7), indicating
that EpGOBP2 without being exposed to its binding components is unresponsive to
EpOR1. A possible mechanism of EpGOBP2 function in the EpOR1 characterisation
assay could be that a complex is formed between the EpGOBP2 and the ligand, and
this complex interact with EpOR1. This is supported by the decrease in EC50 values
for both geranyl acetate and methyl salicylate binding to EpOR1. One hypothesis is
that EpGOBP2 might bind the ligands and release them at the membrane surface to
increase the local concentration of ligand in close vicinity of the OR. It could also be
that the observed decrease in EC50 is a result of EpGOBP2 acting as an activated
ligand, as observed for the Drosophila OBP LUSH, which upon binding its ligand,
11-cis vaccenyl acetate undergoes a conformational change (Laughlin et al., 2008).
LUSH alone in this altered state is able to bind to and activate pheromone sensitive
neurons, indicating the role of a PBP as an activated ligand. EpGOBP2 upon binding
to geranyl acetate and methyl salicylate might become activated and act as a ligand
itself and interact with the OR, hence leading to lower EC50 values.
The combined effect of either ligand binding to EpOR1 in the presence of both
DMSO and EpGOBP2 is similar to each binding EpOR1 in the presence of EpGOBP2
alone. These data indicate that the change in the EC50 values of EpOR1 binding to
methyl salicylate and geranyl acetate could be attributed to the presence of EpGOBP2

The roles of Epiphyas postvittana GOBP2 in odour detection 77
in the assay. Thus raises the possibility that it could be acting as a solubiliser as well
as an activated ligand to confer increased sensitivity on EpOR1.
EpGOBP2 seem to have increased selectivity of EpOR1 to geranyl acetate, as a 100-
fold difference in EC50 value was observed upon replacement of DMSO with geranyl
acetate. In comparison, the EC50 of EpOR1 for methyl salicylate decreased by only
ten-fold. This suggests that EpGOBP2 increases the selectivity of EpOR1 to geranyl
acetate over methyl salicylate. However, this change is not sufficient to make the
assay system more sensitive for geranyl acetate over methyl salicylate, as the EC50 of
EpOR1 for methyl salicylate is still a 1000-fold more than for geranyl acetate. Unlike
PBPs, which can have a significant impact on ligand specificity of PRs when organic
solubilisers are replaced by the PBPs, for example, the response of BmOR3 to
bombykal is obliterated when DMSO is replaced with BmPBP1; EpGOBP2 has a
broader range of ligands and does not have a significant impact on ligand specificity
(Große-Wilde et al., 2006). This may be attributed to moths having a similar number
of PBPs as the number of components that make up the sex pheromones, hence the
ratio of PBP:pheromone component is anywhere between 1:1 or 2:1, thereby allowing
the PBP to be selective for its ligands. On the other hand, only two families of GOBPs
have been identified in moths so far (GOBP1 and GOBP2), while the receptive range
of moths for plant volatiles can be in the hundreds. Hence the need for GOBPs to bind
a broad range of odorants is inevitable if the in vitro suggested roles in this discussion
are applied in vivo by the moth. GOBPs thus become biologically relevant for the
moth‟s success in detecting host plants. Specificity for pheromone detection has been
shown to be coded by PBP/PR/pheromone combination. No such evidence for plant
volatile specificity can be deduced within the scope of this study. Thus EpGOBP2
either acts as an activated ligand itself when bound by odorants, or acts as a
solubilising agent that forms a complex with the ligands and aids its transport and
release near to the membrane surface to increase the local concentration of the ligands
near the receptor. The sensitivity of methyl salicylate and geranyl acetate binding to
EpOR1 was increased in the presence of EpGOBP2, indicating the involvement of
EpGOBP2 in odorant binding by EpOR1. A similar approach can perhaps be used for
characterising ORs in heterologous expression systems which are sensitive to organic
solvents where the organic solvent used for solubilising odorants can be replaced by
binding proteins.

The roles of Epiphyas postvittana GOBP2 in odour detection 78
This research has demonstrated the role of EpGOBP2 as a solubilising agent for plant
volatiles and shown that it can be used to reconstitute a Sf9 cell assay of EpOR1. This
paves the way for further studies where the sensilla lymph can be replicated in tissue
culture. The OR has been expressed in vitro, a role for the GOBP has been
demonstrated, and an ODE can be added to this existing system to complete the
sensilla lymph replica in tissue culture. Such a system would give further insights into
the roles of other players in the moth olfactory system. Recent research has
demonstrated that B. mori GOBP2 binds the sex pheromone components of B. mori,
raising the question of a possible role of moth GOBPs in pheromone reception (Zhou
et al., 2009; He et al., 2010). However, B. Mori GOBP2 is not expressed in
pheromone responsive sensilla (Maida et al., 2005). EpGOBP2 can be further tested
with the sex pheromone components of E. postvittana to confirm whether a
pheromone binding function is unique to the Bombycidae or has been conserved in the
Lepidoptera.

4.1 Introduction
4
Identification of
putative odorant
receptors from Epiphyas
postvittana
The significant role that several members of the order Lepidoptera play in agriculture
as pests has sparked great interest in studying their olfactory system as this is essential
for their detection and location of mating partners and host plants. ORs play a pivotal
role in the recognition of different odorants and their activation results in neuronal
signals which the insect‟s brain interprets (refer to section 1.5.2 for an overview).
Decoding the receptor repertoire of the olfactory system will enable the identification
of specific receptor–ligand combinations of functional importance to various species
of moth. Several different approaches can be employed in the isolation of ORs and
PRs. These approaches are discussed below.
4.1.1 Transcriptome sequencing – EST approach
A library of cDNAs, synthesised from RNA typically from a specific tissue, is made
by shotgun cloning of cDNAs into a plasmid vector, followed by transformation into
suitable host, selection of clones, plasmid isolation and sequencing. A high proportion
of DNA in higher organisms is non-coding, with only a small proportion involved in

Identification of putative odorant receptors from Epiphyas postvittana 80
the coding RNA and protein (Sterky and Lundeberg, 2000) and the references within).
Therefore, sequencing of cDNA, which is transcribed from mRNA eliminates the bulk
of the non-coding sequences.
The differing expression levels of genes can be problematic when identification of
lowly expressed genes is desired. cDNA sequencing can result in many copies of the
highly expressed genes being sequenced and few or no copies of the lowly expressed
genes. One way to mitigate this problem is to normalise the expression levels of all
genes in a cDNA sample by reducing the levels of highly expressed genes. A method
developed by Zhulidov et al. (2004) and Zhulidov et al. (2005) permits the
identification of rare transcripts in a cDNA population. Briefly this method utilises the
specificity of duplex-specific nuclease (DSN) for double stranded DNA as a substrate.
Double stranded cDNA (dsDNA) is synthesised from mRNA of interest by using
specific adaptors. The dsDNA is then melted and allowed to rehybridise under
controlled conditions. A higher tendency of abundant transcripts to rehybridise is
observed compared to rare transcripts. The dsDNA and single-stranded DNA
(ssDNA) population is then treated with DSN, which specifically targets dsDNA for
degradation leaving behind a population that has normalised levels of transcripts. PCR
is then used to generate dsDNA from the ssDNA (Zhulidov et al., 2004; Zhulidov et
al., 2005).
4.1.2 Genome sequencing
Genomic sequencing provides sequence data for all the genetic information contained
within the cells of the organism of interest. It is very useful in identifying the genes
contained within an organism regardless of the expression level of the gene (Bork et
al., 1998). However, gene prediction becomes a challenge as coding regions of genes
are flanked by introns and repeats (Gilbert, 1978). The challenge in genome
sequencing is not obtaining the sequence data itself but assembly of the data and
accurate gene prediction. Most eukaryotic cells have two sources of DNA, the nucleus
and the mitochondria. A larger quantity of mitochondrial DNA (mtDNA) than nucleic
DNA is found in cells hence when isolating genomic DNA for sequencing and
identification of nuclear genes, it is important to reduce the mtDNA content of the

Identification of putative odorant receptors from Epiphyas postvittana 81
total DNA sample. Two broad approaches have been taken to sequence genomes, a
shotgun approach or a clone-by-clone approach.
In the shotgun approach, the genomic DNA of interest is fragmented, then size
selected for library construction (Green, 1997; Weber and Myers, 1997). The ends of
the DNA are end repaired followed by selection of fragments of a certain size class
which are cloned into plasmid vectors and sequenced. The inserts, depending on the
plasmid used can range anywhere from 2 kb to 200 kb. In the clone-by-clone
approach, large overlapping clones (insert size greater than 50 kb) that cover the
whole genome are constructed usually with Bacterial Artificial Chromosomes (BACs)
(Olson et al., 1989; Kim et al., 1996). These BACs or libraries are then individually
fragmented and sequenced by shotgun cloning, after which each BAC is assembled
locally. The individual BACs are then assembled to get the genome sequence of the
organism. This is helpful when assembling large eukaryotic genomes with repeats
with limited computational power.
4.1.3 Recent progress in sequencing field
A turning point in the sequencing field came from the development of low-cost high-
throughput sequencing machines. Several sequencing platforms are now available for
generating millions of bases at a fraction of the price of traditional Sanger sequencing
methods, although the low cost is offset by the generation of only short read lengths, a
limitation of new sequencing technologies. Intense computer power is required for the
correct assembly of thousands of short reads, yet another limitation of new sequencing
technologies. Of the available sequencing platforms of Roche 454 (Ronaghi et al.,
1996; Dressman et al., 2003; Margulies, 2005), Illumina Genome Analyzer (Adessi,
2000; Fedurco et al., 2006; Turcatti et al., 2008), ABI/Solid (Shendure, 2005;
McKernan et al., 2006) and Helicos (Braslavsky et al., 2003; Harris, 2008) the first
two platforms have been used for sequencing the E. postvittana antennal
transcriptome and genome respectively, and will be discussed further.

Identification of putative odorant receptors from Epiphyas postvittana 82
4.1.3.1 454 Sequencing
The Roche 454 sequencing platform uses a sequencing-by-synthesis approach
(Ronaghi et al., 1996; Dressman et al., 2003; Margulies, 2005). dsDNA is
fragmented, then hydrolysed to give ssDNA and adapters are ligated to both ends of
the fragments. Each fragment is then attached to a streptavidin bead via biotin. The
bead is then captured in a droplet that has the necessary nucleotides and buffers for
amplification to take place. Each bead with approximately 10 7 copies of the original
fragment is then deposited into a well of a picotitre plate. The wells‟ dimensions are
such that each one can accommodate only a single bead, together with the reaction
mix. Pyrosequencing of all the wells occurs in parallel, with the release of inorganic
pyrophosphate detected by chemiluminescence, the intensity of the light indicating the
number of each nucleotide incorporated. This being an estimated value gives rise to
slight errors in the sequence reads.
4.1.3.2 Illumina Genome Analyzer
The Solexa technology by Illumina also uses sequencing-by-synthesis where the
fragmented, adaptor ligated ssDNA is attached to the surface of a flow cell which has
a dense lawn of primers that bind to the adaptor sequences (Adessi, 2000; Fedurco et
al., 2006; Turcatti et al., 2008). Solid phase bridge amplification is initiated by the
addition of nucleotides and enzymes, and the fragment bends over as its
complementary strand is synthesized. The amplification reactions result in cluster
formation, each cluster having 1000 clonal copies of each fragment. 40 million
clusters are generated in the amplification in each flow cell which are then sequenced
in parallel to give reads between 35 and 75bp. This is an effective system but a
drawback is the relative short reads produced.
4.1.3.3 Bioinformatics
With billions of bases generated from the new sequencing platforms each day, the
need for improved, powerful computational resources is inevitable. The bioinformatic
analysis following sequence data generation can be divided into the following four
processes; assembly, annotation, gene sequence identification and function prediction
of the proteins (Sterky and Lundeberg, 2000). The major target of the assembly
process is to decrease the number of fragments generated by the sequencing

Identification of putative odorant receptors from Epiphyas postvittana 83
technology by merging the short, overlapping fragments into longer, meaningful
consensus sequences. The challenge in assembling millions to billions of short reads
is the detection and discrimination of sequence ambiguity from regions of repeats.
Due to the short read length and error associated with the pyrosequencing, Sanger
assembly tools cannot be used for assembling these high throughput sequence data
(Ronaghi et al., 1996). Many different assemblers are been designed to overcome the
bottleneck between sequence data generation and analysis of these data to form a
meaningful assembly. The Roche 454 sequencing platform comes packaged with
Newbler for de novo assembly of data generated using this platform (www.roche-
applied-science.com). This assembler can be used for de novo assembly, as has been
shown for bacteria, if sufficient sequence coverage (25 to 30 times) is achieved (Poly
et al., 2007). Short oligonucleotide analysis package (SOAPdenovo) is specifically
designed for assembling Illumina reads (soap.genomics.cs.org.cn). Some other
programs that have been designed for de novo assembly of 30 to 40 bp fragments are
SSAKE (Warren et al., 2007), VCAKE (Jeck, 2007) and SHARCGS (Dohm et al.,
2007). These utilise a greedy approach, whereby reads are chosen to form seeds and
these are extended in both directions by merging overlapping reads into the seeds. The
drawback of such an assembly process is the short length of the contigs generated.
Fragmented assembly and sequencing errors become a problem in gene annotation of
contigs. Sequence alignment with homologous genes from other organisms can be
used to annotate genes or parts of genes even if the full length of the gene cannot be
predicted due to sequencing errors or in-frame stop codons (Pop and Salzberg, 2008)
and the references within). Open reading frames (ORFs) of 300 bp or more can be
identified as potential genes and annotated to some extend by comparing to
homologous genes in databases. The function of a potential gene can be predicted
from sequence similarity in homologous genes, however, specific functional studies
still have to be conducted in order to confirm the function of these predicted genes.
4.1.4 Odorant Receptor Identification in Moths
The first moth odorant receptors were identified in H. virescens through BLAST
search of Heliothis low coverage genomic sequence with candidate OR sequences
from Drosophila used as queries (Krieger et al., 2002). The genomic sequences
obtained from the BLAST search were used as probes to obtain the exonic regions of

Identification of putative odorant receptors from Epiphyas postvittana 84
the sequences from a H. virescens cDNA library. This identified nine OR genes in H.
virescens and further screening of the genomic sequences as well as transcriptome
sequencing have led to the identification of a total of 21 ORs in H. virescens so far
(Krieger et al., 2004). ORs have also been identified from transcriptome sequences of
A. polyphemus, A. pernyi, and M. sexta. In B. mori, 68 ORs have been identified from
transcriptome and genomic sequences, the highest number of ORs identified in a moth
so far (Krieger et al., 2005; Nakagawa et al., 2005; Wanner et al., 2007; Tanaka et al.,
2009). Degenerate PCR has also been used to identify moth ORs, for example,
Mitsuno et al. (2008) identified 10 ORs from three different moth species using
degenerate PCR. However, due to the highly divergent nature of ORs, with only 10-
75% amino acid sequence identity between and within them, degenerate PCR has
been least successful in identifying new ORs, indicating the need for more high
throughput sequencing in order to identify more members of this highly divergent
group.
From glomeruli studies in mammals, it has been observed that each OSN very likely
express only a single OR. OSNs expressing the same OR are scattered throughout the
epithelium, however they merge to the same glomerulus in the olfactory bulb. Hence a
close estimation of the number of ORs in a given organism can be predicted from the
number of glomeruli it possess. Glomeruli studies in tortricids have shown the
presence of anywhere between 48 and 72 glomeruli (Masante-Roca et al., 2005;
Varela et al., 2009). No glomeruli studies have yet been done in E. postvittana so the
number of ORs could be anywhere between 48 and 72. From an EST library of male
E. postvittana antennae, three ORs have been identified (Jordan et al., 2009) (refer to
section 1.7 for more details). ORs and PRs are expressed at low levels in tissues
therefore are under-represented in EST collections, especially in “shallow” Sanger
based collections.

Identification of putative odorant receptors from Epiphyas postvittana 85
4.1.5 Aims
The aim of this research chapter is to increase the receptor repertoire of E. postvittana.
Does E. postvittana contain a similar set of ORs and PRs compared with B. mori in
terms of the number and sequences of ORs, that is, are the E. postvittana ORs one-to-
one orthologs of the B. mori ORs; or are the Tortricidae ORs distinct and different
from those of the Bombycidae, having radiated independently? Based on the 65%
amino acid identity (90% similarity) of BmOR49J and EpOR3, we might expect other
E. postvittana ORs to follow a similar pattern. Does E. postvittana PR also fall in the
PR clade, as has been shown for some other PRs? Do some E. postvittana ORs show
no sex-bias expression while some are more highly expressed in males and some in
females, as is the case with B. mori ORs?
To address these questions multiple approaches are undertaken, each with increasing
power to isolate ORs and PRs. First further screening of the Sanger 1800 ESTs is
done to identify potential PRs by differential screening with male and female antennal
mRNA on a microarray chip. Such an approach will allow the identification of
differentially expressed genes even if their sequences are only represented by 3‟UTRs
on the array. An attempt is be made at designing degenerate primers to the PR clade
of moths and attempting to amplify homologous genes from E. postvittana antennal
cDNA. If the above does not result in the identification of more receptors, then high
throughput sequencing of E. postvittana will be conducted. ORs are expressed at very
low levels so even deep transcriptome sequencing might not identify them all. It is
hypothesized that normalisation of cDNA before sequencing increases the chance of
identifying lowly expressed genes. Further ESTs will be obtained from transcriptome
sequencing of male antennal cDNA and genomic DNA will also be sequenced for
potential ORs using a low coverage whole genome sequencing method.

Identification of putative odorant receptors from Epiphyas postvittana 86
4.2 Materials and Methods
4.2.1 Materials
All the PCR reagents were from Invitrogen unless otherwise stated.
4.2.2 Total RNA Extraction
RNA was extracted from two pools of 100 antennae pairs each for male and female E.
postivttana and from two pools of moth female body without the head, with three
bodies per pool. TRIzol Reagent (Invitrogen) was used for isolating RNA following
manufacturer‟s instructions, and the resulting pellet resuspended in 20 µL of TE
buffer. The concentration of the samples was measured on a nanodrop at an optical
density of 260-280 (OD260/280) (Thermo Sceintific) and the samples stored at -80°C
until use.
4.2.3 mRNA Isolation
mRNA from male and female E. postvittana antennae was isolated using the Ambion
MicroPoly(A)Purist Kit (Applied Biosystems) and the quality assessed on an
Agilent 2100 Bioanalyzer, following manufacturer‟s instructions.
4.2.4 cDNA Synthesis
cDNA was synthesised from 1µg of RNA using either Superscript III reverse
transcript (Invitrogen) or the iscript cDNA synthesis kit (Bio-Rad), following
manufacturer‟s instructions. A no reverse transcriptase control was included for every
sample, and the synthesised cDNA stored at -20°C until required.
4.2.5 Degenerate PCR
To identify conserved regions between E. postvittana OR1 and ORs from three other
insect species belonging to the same phylogenetic clade (M. sexta, B. mori and H.
virescens), multiple sequence alignment of the following OR protein sequences were
conducted using clustalX 1.81 (MsOR1, BmOR3, HvOR6, HvOR16, HvOR14,

Identification of putative odorant receptors from Epiphyas postvittana 87
HvOR15, HvOR11, BmOR5, BmOR7, HvOR13, BmOR4, BmOR1, EpOR1,
BmOR6) with default settings. Two regions each containing the highest levels of
conservation within the alignment were selected to design outer and inner nested
degenerate primers for degenerate PCR using the Ro and Ri primers, given in Table
4.1 (See appendix B for sequence alignment).
Table 4.1: Outer and nested degenerate primers used in degenerate PCR of male E.
postvittana antennal cDNA.
Primer Name Primer Sequence (5’ – 3’)
Outer ARNYTNATHGAYGCNGTNTA
Nested GGNYTNCCNTGGGARTRYATGGA
Ro (outer reverse) ATCGATGGTCGACGCATGCGGATCC
Ri (nested reverse) GGATCCAAAGCTTGAATTCGAGCTC
Five microlitres of ten-fold-diluted cDNA was used as template in the first PCR
together with 1x PCR buffer including 1.5 mM magnesium, 0.2 mM of each dNTP, 2
units of Taq DNA polymerase and 0.5 µM of each outer primer. The final volume was
made up to 50 µL with sterile water. The PCR cycling conditions were as follows:
initial denature at 94°C for 2 minutes, followed by 35 cycles of 94°C for 20 second,
55°C for 30 seconds and 72°C for 1 minutes, and a final extension at 72°C for 10
minutes. One microlitre of product from the first round of PCR was used as template
in a second round of PCR using the nested primers. The same PCR conditions were
used as in the first round of PCR. 10 µL of PCR product was analysed on 1% agarose
gel with 1x SYBR safe DNA gel stain (Invitrogen) and visualised on the ImageQuant
300 system (GE Healthcare Life Sciences).
4.2.6 Cloning and Sequencing
Products greater than 500 bp were excised from the gel and DNA extracted using the
gel purification kit (Qiagen), following manufacturer‟s instructions, after which they
were ligated into pGEM-T Easy vector (Promega) using T4 DNA ligase following
manufacturer‟s instructions. The plasmids were transformed into chemically
competent DH5α cells. Briefly, 2 µL of the plasmid was mixed gently with 25 µL

Identification of putative odorant receptors from Epiphyas postvittana 88
competent cells in a 1.5 mL microcentrifuge tube and incubated on ice for 30 minutes.
The cells were heat shocked at 42°C for 1 minute, and immediately placed on ice.
After three minutes on ice, 225 µL Luria Broth media was added to the tubes and left
to incubate for one hour at 37°C with shaking at 150 rpm. Fifty microlitres of media
solution was then plated on LB agar plates containing 100 µg/mL ampicillin and 40
µg/mL X-galactose (for blue/white screening). After overnight incubation at 37°C
white colonies were screened by PCR with plasmid specific M13 primers by
transferring a small portion of the colony with a pipette tip and dipping it in a PCR
reaction mix containing 1x PCR buffer including 1.5 mM magnesium, 0.2 mM of
each dNTP, 2 units of Taq DNA polymerase and water up to 20 µL. The PCR cycling
conditions were as follows: initial denature at 96°C for 2 minutes, then 30 cycles of
94°C for 30 seconds, 58°C for 30 seconds, 72°C for one minute and a final extension
of 72°C for 10 minutes. The resulting products were visualised on agarose gel as
before and colonies containing an insert greater than 500 bp were picked for
sequencing. Briefly, the colonies were grown in 10 mL LB media supplemented with
100 µg/mL ampicillin overnight at 37°C in a shaking incubator at 220 rpm. The
plasmid was isolated using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced in
both directions using BigDye TM Terminator Version 3.1 Ready Reaction Cycle
Sequencing kit (Applied Biosystems) with capillary analysis performed on the
ABI3730 DNA Analyzer (Applied Biosystems) at the Allan Wilson Centre
Sequencing Services, Palmerston North. The sequences obtained were analysed in
Sequencher v4.2 software (Gene Codes) by aligning both the sequence strands and to
confirm that the overlapping regions matched. These sequences were then searched
against all the available sequences in the NCBI BLAST server (Altschul et al., 1990)
using the tblastx algorithm to identify homologous genes and confirm the identity of
the cloned genes.
4.2.7 Microarray Screening
For differential microarray screening of the male antennal EST library to identify
potential PR candidates, each of the 4472 male antennal EST oligonucleotides were
checked manually to see if the oligonucleotide was in an acceptable region of the
sequence trace file. Candidates for spotting onto a microarray chip were selected
based on their trace file being good and the putative function of the EST from

Identification of putative odorant receptors from Epiphyas postvittana 89
homology searches in NCBI databases yielding proteins with olfactory function or for
whom a function could not be determined. 1809 candidates were selected for which
oligos were designed, synthesized and spotted onto a microarray chip for
hybridisation screening with male and female E. postvittana antennae mRNA. 250 ng
of male and female antennae mRNA each, were labelled with both Cy3 and Cy5 to be
used in a dye swap hybridisation (Cy3 male vs Cy5 female and Cy3 female vs Cy5
male). The microarray hybridisation of the oligonucleotides spotted on the chip with
male and female antennae mRNA was performed at the microarray facility at Plant
and Food Research, Mt Albert, Auckland, as outlined in Janssen et al. (2008).
4.2.8 Microarray Data Analysis
The fluorescence scores from the microarray hybridisation were checked for male-
biased expression and confirmed by checking the corresponding spots by eye. An
oligo was considered to have male antennae biased expression if the same results were
obtained for the dye swap. Genes with oligos showing differential (if fluorescence for
male was ≥ to 2 times that of female) expression were further tested for male-biased
expression by qRT-PCR. The primer design, primer testing and qRT-PCR is outlined
in sections 4.2.12 – 4.2.14 and the primer pairs are given in Table 4.2.
4.2.9 Transcriptome Sequencing
5 µg of male E. postvittana antennal total RNA with an OD260/280 of 1.96 was sent to
Evrogen (www.evrogen.com) for synthesising directionally normalised cDNA, using
the SMART approach and the enzyme DSN, as outlined in section 4.1.1. The
normalised sample was then sequenced on the Roche 454 GS FLX Titanium at the
Department of Anatomy and Structural Biology at The University of Otago. The
sequence data was assembled using the Newbler assembler by the Bioinformatics
department at Plant and Food Research Ltd, Mt Albert, Auckland. The contigs and
singletons were deposited into BioView, a privately held database at Plant and Food
Research Ltd. B. mori OR amino acid sequences were used to perform tblastn
searches of the E. postvittana Insect Database (containing the male antennal contigs
and singletons) and best hit ESTs with e-value less than 0.5 were taken as significant
and selected for further analysis.

Identification of putative odorant receptors from Epiphyas postvittana 90
4.2.10 Genome Sequencing
For genomic sequencing of E. postvittana, the genomic DNA (gDNA) was extracted
from purified nuclei. This was done to reduce the amount of mitochondrial DNA
(mtDNA) being sequenced. The nuclei was isolated first before gDNA extraction as
outlined in section 4.2.11. Library construction (using the Paired-End DNA sample
prep kit VI from Illumina), cluster generation and genomic analysis was carried out at
the Allan Wilson Centre for Genome Sequence (AWCGS, Palmerston North). A total
of 8 lanes of 75 bp paired-end genome sequencing were done on the Illumina 1G
Genome Analyzer (Illumina) at AWCGS. Pipeline analysis was done at the
Bioinformatics department at Plant and Food Research Ltd by Ross Crowhurst. The
sequence data was assembled using SOAPdenovo assembler (Li et al., 2008; Li et al.,
2009). Resulting scaffolds were deposited into the genome server held at Plant and
Food Research Ltd. The putative OR sequences identified from the transcriptome
data, together with known B. mori OR sequences (Krieger et al., 2005; Nakagawa et
al., 2005; Wanner et al., 2007) were used for searching the genomic scaffolds for their
E. postivttana homologues using tblastn. Scaffold hits with e-value less than 0.5 were
taken as significant.
4.2.11 Nuclear DNA isolation and mitochondrial DNA
contamination test
[This protocol has been modified from the method by (Guo; Lopez-Gomez and
Gomez-Lim., 1992)].
4.2.11.1 Isolation of insect nuclei
Refer to Appendix C for buffer recipes used in this section. Three grams of female E.
postvittana pupae were ground in a mortar and pestle in liquid nitrogen and
transferred to a beaker with 300 mL of ice-cold nucleic extraction (NEB) complete
buffer. The resulting homogenate was filtered through 4-6 layers of cheesecloth into a
sterile glass beaker on ice. The filtration step was repeated through 2-4 layers of
miracloth into a 500 mL sterile glass cylinder. The volume was adjusted to 294 mL
with NEB complete buffer and 6 mL of 25% Triton X-100 in NEB complete buffer.
The cylinder was sealed with parafilm and mixed very gently by inversion 10-20

Identification of putative odorant receptors from Epiphyas postvittana 91
times. The homogenate was divided into six 50 mL Falcon tubes and spun down at
588 rpm at 4-10°C, for 2 minutes. The supernatant was transferred to a new set of 50
mL Falcon tubes and centrifuged at 3300 rpm, 4-10°C for 15 minutes. A reddish
pellet was clearly visible. The supernatant was transferred to the waste container and
each pellet resuspended with 50 mL NEB– no -mercaptoethanol and mixed gently
by inversion until the pellet was resuspended. The samples were spun down as before
and the supernatant transferred to the waste container. Each pellet was resuspended
with 5 mL NEB– no -mercaptoethanol and all pellets collected into one weighed
Falcon tube. More NEB– no -mercaptoethanol was added to a final volume of 50
mL, spun down again as before and the supernatant transferred to waste container.
The tube was weighed to find out the total amount of nuclei isolated and the tube kept
on ice.
4.2.11.2 Genomic DNA extraction
0.2 grams of nuclei pellet was resuspended in 14 mL lysis buffer and mixed by
inversion. 14 µL of RNase A 50 mg/mL (Qiagen) was added and mixed by inversion,
followed by incubation at 37°C with gentle shaking for 10 minutes. 1.4 mL 5 M
potassium acetate pH 7 was then added to it followed by addition of 3.5 mL 100%
ethanol. The sample was vortexed at maximum speed for 30 seconds and extracted
with equal volume of chloroform:isoamyl alcohol (24:1). The tube was then placed
horizontally in a platform shaker at room temperature and shook gently for 5 minutes.
The sample was spun down at 3000 rpm for 10 minutes at room temperature and the
supernatant collected and placed in a new 50 mL Falcon tube. Equal volume of ice
cold isopropanol was added to the sample and mixed by inversion, followed by
incubation at -20°C for 30 minutes. The sample was spun down as before, the pellet
washed with same volume of 70% ethanol. The DNA pellet was air dried at room
temperature for 20-30 minutes and resuspended in 100–200 µL TE buffer pH 7.5. The
DNA quality was checked at OD260/280 on the nanodrop (Thermo Scientific).
4.2.11.3 Mitochondrial DNA contamination test
A ten-fold serial dilution of the gDNA was made from 1 in 10 to 1 in 100 million with
TE buffer. The primer pairs MT6 forward 5‟-
GGAGGATTTGGAAATTGATTAGTTCC-3‟ and MT9 reverse 5‟-

Identification of putative odorant receptors from Epiphyas postvittana 92
CCCGGTAAAATTAAAATATAAACTTC-3‟, specific for the cytochrome oxidase I
gene of the mitochondrial genome of E. postvittana, were used in a PCR reaction to
test for mtDNA contamination in the gDNA sample. Primers for Takeout 3, which is a
nuclear gene, were used for a direct comparison of the nuclear versus mtDNA (5‟-
AAAGGCGAGGGTCACTACAAG-3‟ TO3 forward primer and 5‟-
GGTTCTTCAGTGCGTACACCT-3‟ TO3 reverse primer). The PCR reaction mix
was prepared and the first round of PCR conducted, as stated in section 4.2.5. The
products were analysed on 1% agarose gel with 1x SYBR safe DNA gel stain
(Invitrogen) and visualised on the ImageQuant 300 system (GE Healthcare Life
Sciences). One lane of 75 bp paired-end genome sequencing was done as stated in
section 4.2.10 and Bowtie, a short read aligner (Langmead et al., 2009) was used for
mapping mitochondrial sequences in GenBank and a 2,216 base pair region of the E.
postvittana mitochondrial genome containing the cytochrome oxidase I and II genes
to the 13 million paired-end reads obtained from one lane of Illumina sequencing.
4.2.12 qRT-PCR primer design
Primers were designed using Oligo Explorer 1.1.0 (Kuulasmaa, 2000) and where
possible primers were designed using the following stringent conditions: optimal
temperature between 50-60°C, GC% content between 40-60%, and at least 3 GC
dinucleotides at the 3‟end for primer stability. The primers were between 20–23 bp in
length and the amplicon size was between 100–200 bp. The primers were made such
that the formation of primer dimers are minimised and the primers do not self anneal.
The primer pairs for the candidate OR sequences identified from the microarray
screening and transcriptome sequencing are given in Table 4.2.

Microarray OR
454 Transcriptome OR candidate ESTs
Identification of putative odorant receptors from Epiphyas postvittana 93
Table 4.2: Primer pairs for qRT-PCR amplification of products from OR candidates
identified from microarray screening and 454 transcriptome sequencing of male E.
postvittana antennae.
candidate ESTs
EST Number Primer Forward (5’ – 3’) Primer Reverse (5’ – 3’)
23893 AGGCATCACCAAAGGTAAG CGATAGAAACGCTGTCATTG
25116 GTAATACGCCTTCTGAATGC CCAAAGCGTATCAAAGTTCC
25174 GCAACTCATCAGAAAACTCG AGACAGCAGAGTGAAATCCAG
25214 GCCCCTTGTATTATTATTGGTC AGATAGGTCTGCCTTTTACACG
1032444 CACTGCGGGTATGTTTCCTG TACCCTGCCCTTGATTTCG
1037459 CGTGCTCATCCAAAGATCC ATGCCTTGCCGAAGTCATC
1051140 GCGAGCTGCGTAAAGTTGG TGGAATACAGGGGTCTGGTC
1041667 CTCGGCAACAAATAAAACGC ACCATTTGCTCTCATAAGGC
1043845 AGACGCAGCGTACAAAAGC AATAACCCATGGCAGACAAC
1042257 TTTATTCGGGGCACAAACG GGATTCCGCTGAAAGGATTC
1034291 AACTGGGCTTAAGTAAGTGC TAGCCCTGGAATGAAACAC
1037304 ATGCCTTTCGCTTTGATGC TCCCCACTTGCTATCGTAAC
1040652 CGAGACGTTTTCTTGTGTC ATAGGTCAGCCGACTGTAG
1034929 ACTACGATGCCACAGAAAGG GCCCAGAGGTAGCGATATG
1218954 TCCGTTTCCTTGACATAGG ATTGAGGCGAGGCTGATAG
1212362 TCAATGGGGGGAAGTAATAG TGAGCTGCGGTATATTTCTG
1209721 TTGGGAGTCGACATCTGTG CCACCGACATTGTTTTCTGC
1037495 CCAATACAATCAGGTGAAGC CGTTGTCTGTCGGTCTTAG
1234206 CAAACGCCCCAGTACCAAG CTCTCGCTCACCACTTTCAC
1233473 TAGCACGCTGACATTGATG TACTTCACTGGTTCTGTGGC
1039296 AACGAGGTTGCTGTTGAAG ACATACGCGCCTTACTACG
1212480 CAAACGGCATCCAAGAACTG GTGACGGTCACCACAACAC
1207127 GGACGAGTCACTGAACTGC CGGGATACCCATTAGAGAG
1226006 GCCCTTCTCTACACTCACTG CTGCGTCTAATCTTCGTTG
1043233 CTTCCCCAAGATGAAATGCC GACCCCTAACCCAACTGATG
1201411 TGGCTTCGCTTATATGGAC CCCTAATGGTGGTTGATGG
1049412 AGGTCTGGCAATCATCTCAG GGTGCCGTGTCTTATTATG
1041985 GCTCCAAGGTGAGTACAAC CTGGCTGAGCTAAATCTGC
1046227 ACCAAGGCCAAAGAGAGAAC CTTCGACTGGCAATAAGGTG
1039491 GCCAGCTGCACTCATCAAG GCTGGCGATTCTCCTATTC

Identification of putative odorant receptors from Epiphyas postvittana 94
4.2.13 qRT-PCR primer test
Each set of primers were used with the cDNA samples from the three different tissues
in a gradient cycler to optimise the PCR conditions and obtain the optimal annealing
temperature for each. The standard PCR reaction components were as follows: 1x
PCR buffer including 1.5 mM magnesium, 0.2 mM of each dNTP, 2 units of Taq
DNA polymerase, 0.5 µM of each primer, 30 ng cDNA template and water added to a
reaction volume of 20 µL. The PCR cycling conditions were as follows: 95 o C for 2
minutes, followed by 40 cycles at 94 o C for 15 seconds, gradient from 50 to 60 o C for
20 seconds, 72 o C for 30 seconds and a final elongation at 72 o C for 10 minutes. The
gradient tested was from 50 o C in column 1 through to 60 o C in column 12. PCR
products were visualised on 1% agarose gel with 1x SYBR safe DNA gel stain
(Invitrogen) and visualised on the ImageQuant 300 system (GE Healthcare Life
Sciences).
4.2.14 Quantitative Real-Time PCR
Quantitative real-time PCR were performed in 384 well plates on either Applied
Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems) or on the
Roche LightCycler 480 instrument (Roche, Germany), with SYBR green as the
fluorescent reporter. Each primer template combination was set up in triplicate and a
no template water control was included for each primer pair, (two pools of each
template giving a total of six different templates). The housekeeping genes Ef1α and
α-tubulin (Turner et al., 2006) were used due to their consistent expression in
different E. postvittana tissues.
For qRT-PCR performed on Applied Biosystems 7900HT Fast Real-Time PCR
System, the plate and instrument set up were designed using the SDS 2.1 software
(Applied Biosystems). The assay type performed was absolute quantification followed
by a dissociation curve analysis at the end of the run to check for single PCR
products. The reaction conditions were same as that for primer tests except for the
addition of 0.2 µL of 1/1000 SYBR green dye (Molecular Probes). The PCR cycling
conditions were 95 o C for 2 minutes, followed by 40 cycles at 94 o C for 15 seconds,
55 o C for 20 seconds, 72 o C for 30 seconds followed by the generation of a dissociation

Identification of putative odorant receptors from Epiphyas postvittana 95
curve with the following thermal profile; 95°C for 15 seconds, 60°C for 15 seconds,
95°C for 15 seconds, with a ramp rate of 2%. The result files obtained from the run
were loaded into the SDS 2.1 software and the amplification and dissociation curves
were checked for consistency between the triplicates. Wells that had more than one
peak in the dissociation curve, hence more than one PCR product were omitted from
further analysis. Primer PCR efficiencies were calculated using the LinRegPCR
(Ramakers et al., 2003) program and the Cycle Threshold (Ct) values for all the
samples were converted to quantity of product formed in each sample by the
following equation:
Quantity = (PCR efficiency)
(minimum quantity for a particular treatment – current Ct value)
The mean quantities of the housekeeping genes for each tissue type were used with
the software geNorm (Vandesompele et al., 2002) for calculating the normalisation
factor for the individual tissue types. These normalisation factors were then used for
calculating the relative expression of the samples.
For qRT-PCR performed on a Roche LightCycler 480 instrument, the reaction
mixture consisted of 5 µL 2x FastStart SYBR Green Master (Roche, Germany), 0.5
µM of each primer and 30 ng of cDNA template, in a total reaction volume of 10 µL
made up with nuclease-free water. The thermal profile of the run was as follows: 95 o C
for 5 minutes at ramp rate ( o C/s) of 4.8, then 40 cycles at 95 o C for 15 seconds at ramp
rate of 4.8, 55 o C for 30 seconds at ramp rate of 2.5 and 72 o C for 20 seconds at ramp
rate of 4.8. A melt curve was then generated at the end of the amplification to verify
single products with the following profile: 95 o C for 5 seconds at a ramp rate of 4.8,
65 o C for 1 minute at ramp rate of 2.5 followed by continuous acquisition at 97 o C at a
ramp rate of 0.11. Analysis of the raw data was carried out with the LightCycler 480
software version 1.5 (Roche). PCR efficiencies of the primers were calculated using
the exported amplification data at every cycle using the program LinRegPCR
(Ramakers et al., 2003). The relative quantification of the test genes was calculated
based on relative expression of target versus housekeeping genes, as stated in Pfaffl
(2001).

Identification of putative odorant receptors from Epiphyas postvittana 96
4.2.15 Rapid Amplification of cDNA Ends (RACE)
For each 3‟RACE-PCR reaction, 3 µL of ten-fold diluted male E. postvittana antennal
cDNA was used together with a gene specific forward primer (designed from a known
region on the EST of interest) and the reverse oligo dT primer (RoRidT16) that is
specific for the mRNA poly A + tail. The gene specific forward primers used are given
in Table 4.3. The reaction mix consisted of 1x PCR buffer, 1.5 mM magnesium, 0.2
mM of each dNTP, 2 units of Taq DNA polymerase, 0.5 µM of each primer. The final
volume was made up to 25 µL with sterile water. Touchdown PCR was performed as
follows: initial denature at 94 o C for 4 minutes followed by 20 cycles at 94 o C for 30
seconds, 60-50 o C for 30 seconds and 72 o C for 30 seconds. This was followed by
another 10 cycles at 94 o C for 30 seconds, 50 o C for 30 seconds and 72 o C for 30
seconds. A final extension was carried out at 72 o C for 7 minutes. The products were
analysed on 1% agarose gel. Products greater than 300 bp were selected and
sequenced.
Table 4.3: Gene specific primers used for amplifying 3‟RACE products from OR
candidates from the 454 transcriptome assembled contigs.
Putative OR Gene Gene Specific Primer (5’ – 3’)
EpOR7 GGATCCTAACGCATTGGCAAAGT
EpOR23 GCAGCGTACAAAAGCCTGTGGTA
EpOR24 GAGTGGAATTCGCTCCCATCGTC
EpOR30 TATTTACAGCTTGATCGCTGTCG
EpOR32 AACTTTACGCAGCTCGCCGCCAT
EpOR33 GATACGAGCGACGAGGTCCCTCT
EpOR34 GCTTATTCAGCCTACGCTGGAGA
EpOR39 GGAGGATTGGAGCACACGTATAT
EpOR41 GATGGGGCCTTATGAGAGCAAAT
EpOR48 GACATTTCTTCATAGGTCCGTCG
EpOR49 ATGCGCGAGGACTACAGTCGGCT
EpOR50 GTTTTGATTTGTATCTGTGCGCC
EpOR51 GGAGTTTTAGGGCAAACATAGTT
EpOR52 TGATTGCCGTGCTGCTGGCGATT

Identification of putative odorant receptors from Epiphyas postvittana 97
5‟ RACE was used to isolate the 5‟ ends of the coding regions of the genes given in
Table 4.4, each PR candidate from the microarray analysis and 454 transcriptomics
sampling based on their tissue specific expressions in male E. postvittana antennae (as
shown in Figures 4.1 and 4.4). The 5‟RACE System for Rapid Amplification of
cDNA ends, Version 2.0 (Invitrogen) was employed according to manufacturer‟s
instructions.
First strand cDNA was synthesized from male antennae E. postvittana RNA using the
outer gene specific primers (GSP) given in Table 4.4. This was done for each of
individual ESTs given in Table 4.4 so as to synthesise cDNA specific for these ESTs,
thereby enriching the cDNA for the particular gene. The cDNA synthesis was done
using either superscript III reverse transcriptase (Invitrogen), or with iscript reverse
transcriptase (Bio-Rad) following manufacturer‟s instructions, except that the oligo
dT primer was replaced by the primer GSP. The synthesized cDNA was then purified
using the PCR purification kit (Roche) following manufacturer‟s instructions. The
cDNA was tailed at the 3‟end with Terminal Transferase (TdT) (Roche). This adds a
homopolymeric tail of cysteine residues at the 3‟end of the cDNA hence a primer that
binds to these residues can be used to amplify up the target gene with GSP at the other
end. A 20 µL tailing reaction was carried out as follows: 9 µL purified cDNA, 4 µL of
5x tailing buffer, 2 µL of 10 mM dCTP, 4 µL of 25 mM CoCl2 and 1 µL of TdT
enzyme were mixed and incubated for 1 hour at 37°C. This was followed by heat
inactivation of the TdT at 65°C for 15 minutes. Each of the tailed cDNA were then
used in 5‟RACE reaction together with their respective outer GSPs given in Table 4.4.
A 50 µL PCR reaction mix was setup as in section 4.2.5 with 5 µL of the tailed cDNA
with 0.5 µM of its corresponding outer GSP and 0.5 µM of AAP primer (Table 4.4).
The following PCR cycling conditions were used: 94°C for 2 minutes, followed by 30
cycles of 94°C for 10 seconds, 50°C for 30 seconds, 72°C for 1 minute and a final
extension was carried out at 72°C for 10 minutes. One microlitre of this PCR product
was used as template in a second round of amplification with the nested GSP and
AUAP primer (Table 4.4), using the same reaction components as before. Products
from this second round of PCR amplification were analysed on 1% agarose gel and
products greater than 500 bp were excised from the gel, the DNA extracted and
cloned and sequenced as stated in section 4.2.6.

Identification of putative odorant receptors from Epiphyas postvittana 98
Table 4.4: Primers used for amplifying 5‟ RACE products from the two candidate
PRs identified from microarray screening and the three PR candidates identified from
the 454 transcriptome sequencing of E. postvittana.
EST Identified
from:
Microarray
screening
454
Transcriptome
sequencing
Primer Nucleotide Sequence (5’ – 3’)
25174 (outer) AGATTTCTCTGATTATTTACACAATGTATA
25174 (nested) GTTGCACAGTGGCGACAGACCGGCACTTTT
25214 (outer) TTATTACTAAAATTACCCTCGTAACAACAA
25214 (nested) CCCGAATGATATTCCAAATGCTTACAAACT
1032444 (outer) GTGGCCCAAAAGGCGCAAAGCCCGAAT
1032444 (nested) GCCATAACTTCGATACCCACTGCCCTC
1037459 (outer) GCGGGAACAGCGCGCAGTTCACACAAC
1037459 (nested) GAAGTCATCCCCTTTATGTCGACAGCG
1042257 (outer) GTATGAGTATTGTACCGTTTGTGCCCCG
1042257 (nested) CTTCCAGGCAGACAGCCCTGAACACCG
Abridged anchor primer (AAP) GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG
Abridged universal amplification
primer (AUAP)
4.2.16 Bioinformatics
GGCCACGCGTCGACTAGTAC
The predicted OR proteins from the low coverage genomic scaffolds were translated
into the six reading frames and compared with the B. mori OR exons for determining
the exons of the proteins. The program SplicePredictor
(http://deepc2.psi.iastate.edu/cgi-bin/sp.cgi) was also used for predicting the
exon/intron boundaries. A phylogenetic tree was constructed using the PHYLIP3.6a3
package (Felsenstein, 2005) of all the known and putative E. postvittana ORs together
with the ORs from B. mori (Warren et al., 2007; Tanaka et al., 2009), H. virescens
(Krieger et al., 2002; Krieger et al., 2004), P. xylostella, M. separata, and D. indica
(Mitsuno et al., 2008).
The protein sequence of EpOR34 was submitted to seven different transmembrane
prediction programs including HMMTOP (Tusnady and Simon, 2001), TMPred
(Hofmann and Stoffel, 1993), TMHMM (Krogh et al., 2001), DAS (Cserzo et al.,
1997), SPLIT (Juretic et al., 1993), TMMOD (Kahsay et al., 2005) and TOPPRED
(Claros and von Heijne, 1994). The predicted TM regions were compared and a

Identification of putative odorant receptors from Epiphyas postvittana 99
consensus of the different domains was made manually. A topology map of EpOR34
was then drawn in TOPO2 (http://www.sacs.ucsf.edu/TOPO-run/wtopo.pl) using the
consensus sequence above.
4.3 Results
4.3.1 Microarray Analysis
A microarray analysis of 1809 ESTs from a male antennal cDNA library of E.
postvittana was carried out to identify male-specific genes. From the microarray
screening, four ESTs had more than two times higher expression in male than female
antennae (EST numbers 23893, 25116, 25174 and 25214). The sex-biased expression
of two (ESTs 25174 and 25214) of the four ESTs were confirmed by qRT-PCR
(Figure 4.1).
Figure 4.1: Quantitative RT-PCR of four candidates PRs as identified from a
differential microarray screening of EST oligos. The error bars represent standard
errors of three technical replicates for each tissue type. ND = not detectable.
3‟RACE-PCR was not done on ESTs 25174 and 25214 as the original sequences
already had the poly A + tail sequence. The 5‟RACE-PCR was only able to extend the
original EST sequences by 350–400 bp. Further 5‟RACE to extend the sequences

Identification of putative odorant receptors from Epiphyas postvittana 100
further were unsuccessful. Tblastx of the sequences of the 5‟RACE-PCR products of
ESTs 25174 and 25214 in the NCBI BLAST server did not yield any significant hits.
The original EST sequences were also used to search the blastable E. postvittana 454
transcripts as well as the assembled genomic scaffolds using the tblastx tool. For both
the ESTs, the 454 transcripts hit were the same length and sequence as the original
sequences hence deep transcriptomics was not able to extend these sequences any
further. Fragments of the original ESTs were also found in the genomic scaffolds,
however the scaffolds were not long enough to extend the EST sequences any further.
4.3.2 Deep Transcriptomics
The male E. postvittana antennal cDNA was normalised prior to deep transcriptomics
in an attempt to reduce to levels of highly expressed genes and increase the chance of
sequencing the lowly expressed ORs. The male E. postvittana antennal cDNA
received from Evrogen after normalisation had an average length of 1 kb. Figure 4.2
shows 500ng of the cDNA before (lane 1) and after (lane 2) normalisation. The highly
fluorescing bands in lane 1 seemed to have been evened out after undergoing DSN
treatment suggesting that the cDNA population in the sample had been successfully
normalised.

Identification of putative odorant receptors from Epiphyas postvittana 101
Figure 4.2: E. postvittana male antennal SMART-amplified cDNA before (1) and
after (2) normalisation. Approximately 500ng of cDNA were loaded on the gel. Lane
M is 1kb DNA marker.
4.3.2.1 454 Sequencing Statistics
A single whole plate of 454 titanium EST sequencing of the normalised E. postvittana
male antennae cDNA yielded 1,091,459 EST sequences. This data underwent
validation before any analysis was conducted. The validation process involved
filtering the ESTs and trimming possible vector sequences, removal of low quality
sequences as well as sequences shorter than 50 bp. After the validation process, the
number of ESTs remaining were 903,121 totalling 188,511,228 bp. Contig assembly
was done using Newbler software under default settings. This resulted in 21,627
contigs and 94,579 singletons. The longest contig was 1994 bp and the N50 (the
contig length that produces half the bases in the genome) of the combined contigs and
singletons was 279 bp. An analysis of the 454 sequence data is given in Table 4.5.
The contigs and singletons were deposited into a privately held Bioview Insect
Database at Plant and Food Research Ltd and automatically annotated using Gene
Ontology (GO) analysis.

Identification of putative odorant receptors from Epiphyas postvittana 102
Table 4.5: Analysis of the 454 transcriptome sequence data from the raw data
obtained (before contig assembly) through to the processed data, assembled contigs
and singletons.
Before contig
assembly
No. of
ESTs
Bases (bp) N50 Average
length (bp)
Median
(bp)
Longest
(bp)
903121 188511228 208.7 192 594
Contigs 21627 6884597 369 318.3 311 1994
Singletons 94579 16837407 234 178.0 152 579
Contigs +
singletons
116206 23722001 279 204.1 175 1994
4.3.3 Mitochondrial DNA contamination
Before performing whole genome sequencing, an attempt was made at minimising the
sequencing of mitochondrial DNA thereby increasing sequencing of nucleic DNA (as
the OR genes are nuclear), the nucleic was isolated first, then gDNA extracted from it.
E. postvittana DNA prepared from nuclei contained less mitochondrial DNA than
nuclear DNA based on semi-quantitative PCR of the mitochondrial and nuclear genes
(Figures 4.3). The DNA prepared from nuclei was then used as a template for
Illumina sequencing. Just to be cautious that the gDNA sample has minimal mtDNA
contamination, one lane of Illumina sequencing was initially done on the gDNA.
Bowtie, which is an ultrafast short read aligner (Langmead et al., 2009) was used for
mapping the mitochondrial sequences in genbank and a 2,216 base pair region of the
E. postvittana mitochondrial genome containing the cytochrome oxidase I and II
genes to the 13 million paired end reads. The test parameters for bowtie allows up to
only 3 mismatches. Only 92 out of the 13 million paired end reads were predicted to
be mitochondrial, suggesting that mtDNA contamination is minimal. Another 7 lanes
of Illumina sequencing was then done in order to obtain higher coverage of the
genome and an attempt at assembling longer scaffolds for use as a sequence set able
to be searched using blast.

Identification of putative odorant receptors from Epiphyas postvittana 103
850bp
650bp
1kb+
ladder
Figure 4.3: Semi-quantitative PCR on nuclear (Takeout 3) and mitochondrial
(cytochrome oxidase I) genes using total DNA extracted from purified nuclei as
template. Lanes A to G show PCR performed on a 10 fold serial dilution of the DNA
template from 1 in 100 to 1 in 100 million with E. postvittana Takeout 3 primers.
Lanes H to N are PCR performed on the same template serial dilution with
cytochrome oxidase I primers.
4.3.4 Illumina Sequencing
The assembly of the 8 lanes of Illumina sequencing resulted in 299,968,860 bases
being assembled into 843,963 scaffolds with the longest scaffold being 19,645 bp,
with an N50 of 511 bp. The sequences were deposited into a privately held genome
server at Plant and Food Research Ltd for blast querying and data mining.
4.3.5 Data Mining
Nucleic DNA products Mitochondrial DNA products
A B C D E F G H I J K L M N
Tblastn of the E. postvittana 454 transcripts and genomic sequences with known B.
mori OR sequences resulted in the identification of a further 49 putative ORs,
bringing the total number of ORs identified from E. postvittana to 52. The ORs with
shortest and longest predicted coding region lengths are EpOR49 (40 amino acids)
and EpOR21 (475 amino acids) respectively. The full length of only one putative OR
was identified (EpOR34). The highest level of amino acid identity shared between E.
postvittana and B. mori ORs is 88% between EpOR2 and BmOR2. Interestingly,
EpOR8 shares 71% amino acid identity with BmOR8, a larval-specific OR in B. mori.

Identification of putative odorant receptors from Epiphyas postvittana 104
Table 4.6 summarises the properties of the E. postvittana ORs, with the predicted
coding region length available for each OR, its closest B. mori homologue based on
pairwise distance matrix calculated in ClustalX and the method(s) that identified each
OR. TMPRED was used for predicting the topology of the ORs for which more than
350 amino acids of the predicted coding regions were available. Of these, EpOR2, 32,
34 and 47 were predicted to have intracellular N-terminus (shaded in orange and
purple on Figure 4.4). EpOR1 and EpOR6 share the most amino acid identity with
BmOR1, a PR of B. mori. EpOR1 has been demonstrated to be a receptor for plant
volatiles in Chapter 2; hence EpOR6 (shaded in yellow on Figure 4.4) is predicted to
be a candidate PR of E. postvittana.
Table 4.6: E. postvittana putative OR repertoire to date. The ORs were identified
either by Sanger sequencing (S), 454 transcriptome sequencing (T) or low coverage
whole genome sequencing (G). The B. mori homologues of the corresponding E.
postvittana ORs are given, together with the percentage amino acid identity shared
between the homologues. This data is based on the incomplete sequences of the
putative E. postvittana ORs and can change as the full length sequences become
available.
Putative
OR
Length
(amino
acids)
Closest B. mori OR
identified with the
available length
% amino acid identity with
B. mori ORs with the
current sequence length
Method
EpOR1* 415 BmOR1 38 S, T, G
EpOR2* 474 BmOR2 88 S, T, G
EpOR3* 410 BmOR49J 65 S, T, G
EpOR4 268 BmOR56 60 G
EpOR5 304 BmOR68 22 G
EpOR6 241 BmOR1 17 T, G
EpOR7 308 BmOR4 26 T, G
EpOR8 324 BmOR8 71 G
EpOR9 225 BmOR4 14 G
EpOR10 349 BmOR27 22 G
EpOR11 113 BmOR26 19 G
EpOR12 293 BmOR15 45 G
EpOR13 271 BmOR13 52 T, G
EpOR14 118 BmOR14 20 T, G
EpOR15 319 BmOR13 20 T, G
EpOR16 206 BmOR41 23 G
EpOR17 243 BmOR49 60 G

Identification of putative odorant receptors from Epiphyas postvittana 105
EpOR18 312 BmOR30 37 G
EpOR19 209 BmOR28 17 G
EpOR20 131 BmOR14 37 T, G
EpOR21 475 BmOR49 18 T, G
EpOR22 84 BmOR6 32 T, G
EpOR23 206 BmOR23 33 T, G
EpOR24 114 BmOR24 28 T, G
EpOR25 257 BmOR18 47 G
EpOR26 188 BmOR26 22 G
EpOR27 329 BmOR27 57 T, G
EpOR28 170 BmOR28 15 G
EpOR29 185 BmOR29 57 T, G
EpOR30 257 BmOR30 28 T, G
EpOR31 282 BmOR16 38 G
EpOR32 359 BmOR32 41 T, G
EpOR33 118 BmOR68 22 T, G
EpOR34 395 BmOR33 30 T, G
EpOR35 324 BmOR36 49 G
EpOR36 152 BmOR61 18 T, G
EpOR37 219 BmOR55 43 G
EpOR38 162 BmOR38 55 G
EpOR39 120 BmOR39 60 T, G
EpOR40 113 BmOR16 31 G
EpOR41 210 BmOR29 42 T, G
EpOR42 293 BmOR42 33 T, G
EpOR43 175 BmOR63 22 G
EpOR44 211 BmOR44 29 G
EpOR45 77 BmOR15 22 G
EpOR46 221 BmOR47 17 G
EpOR47 350 BmOR60 54 T, G
EpOR48 97 BmOR52 21 T
EpOR49 40 BmOR30 30 T
EpOR50 70 BmOR46 20 T
EpOR51 76 BmOR26 23 T
EpOR52 59 BmOR64 27 T
*These E. postvittana ORs were identified by Jordan et al. (2009), while all the other
ORs have been identified in this study.

Identification of putative odorant receptors from Epiphyas postvittana 106

Identification of putative odorant receptors from Epiphyas postvittana 107

Identification of putative odorant receptors from Epiphyas postvittana 108

Identification of putative odorant receptors from Epiphyas postvittana 109

Identification of putative odorant receptors from Epiphyas postvittana 110
Figure 4.4: Multiple sequence alignment in ClustalX of E. postvittana ORs identified
to date. Transmembrane positions obtained from consensus of EpOR1, 2, 3 and 34 are
shown as light orange bars above the alignment, with the roman numeral indicating
the TM domain region. The predicted E. postvittana PRs are shaded in yellow, the
ORs with predicted intracellular N-terminus are shaded in purple while EpOR34
which has both these features is shaded in dark orange.
4.3.6 Quantitative Real-Time PCR
The expression levels of 23 of the 49 new putative E. postvittana ORs (these 23
putative ORs were identified from the 454 transcriptome sequencing; the Illumina
sequencing data became available towards the end of the project and were not able to
be analysed by qRT-PCR due to time restriction) together with EpOR1, 2 and 3 were
examined in male antennae, female antennae and female body (minus head) tissues
using qRT-PCR (Figure 4.5). Sex-biased expression was detected in three ORs, with
EpOR30, 33 and 34 being expressed more than 500 times more highly in male than
female antennae, therefore are predicted to be the PRs of E. postvittana (these are
shaded in yellow and orange on Figure 4.4). A further 12 ORs (EpOR7, 13, 23, 24,
36, 39, 41, 47, 49, 50, 51 and 52) had two to ten-fold higher expression in male than
female antennae while seven ORs (EpOR14, 20, 21, 27, 32, 42 and 48) had similar
expression levels in male and female antennae. Expression of eight ORs (EpOR7, 13,
24, 27, 33, 49, 50 and 52) was also detected in the body.

Identification of putative odorant receptors from Epiphyas postvittana 111
Figure 4.5: Expression levels of the putative E. postvittana ORs in male antennae, female antennae and body tissues. The housekeeping genes
Ef1α and α-tubulin were used for normalising the expression levels. The error bars represent standard errors calculated from two biological
replicates. * The tissue expression of these ORs was not determined.

Identification of putative odorant receptors from Epiphyas postvittana 112
A preliminary phylogenetic tree was constructed from multiple sequence alignment of
the 52 E. postvittana ORs together with known ORs from five other moth species B.
mori (Wanner et al., 2007; Tanaka et al. 2009), H. virescens (Krieger et al., 2002,
2004), P. xylostella, M. separata, and D. indica (Mitsuno et al., 2008) (Figure 4.6).
The sequences were obtained from GenBank with accession numbers given in the
mentioned studies. The alignment producing this tree is given in Appendix D. This
tree has been constructed using partial E. postvittana OR sequences therefore the
phylogenetic information presented here may be inaccurate and only the availability
of the full length sequences of these putative ORs will give a true relationship of these
ORs with those from the five other moth species. The male-biased sex pheromone
receptor clade to which all the Lepidoptera PRs identified to date belong to, and the
female-biased clade of B. mori are indicated, together with the highly conserved
Drosophila OR83b clade on Figure 4.6. The putative E. postvittana PRs are shaded in
red. Interestingly, the three sex-biased putative PRs (EpOR30, 33 and 34) cluster
together, leading us to deduce that a different set of ORs (as opposed to the PRs of
other moths forming one clade) have been co-opted in E. postvittana for a role in
pheromone reception.

Identification of putative odorant receptors from Epiphyas postvittana 113
Figure 4.6: A preliminary phylogram resulting from multiple sequence alignment of
ORs and PRs from moths, together with EpORs1–52. The tree was constructed using
Fitch using John-Thorton distances and rooted with the clade of EpOR2 orthologues.
Bootstrap values were calculated from 1000 bootstrap replicates and are given as a
percentage value on the nodes, where possible. The putative E. postvittana PRs are
shaded in red.

Identification of putative odorant receptors from Epiphyas postvittana 114
4.3.7 EpOR34 – a putative PR
The only putative E. postvittana OR for which a full length coding region has been
obtained from the 49 new ORs identified is EpOR34. The predicted coding region of
EpOR34 (Figure 4.7) was assembled from the partial transcript of EST 1032444, a
2033 bp genomic scaffold and 3‟RACE-PCR. The predicted coding region was 1185
bp and encodes 395 amino acids from the start methionine to the stop codon. From the
genomic sequence, two introns were identified, as indicated by red arrowheads in
Figure 4.7. The first intron occurred after amino acid 258 and was 87 bp while the
second intron was after amino acid 291 and was 351 bp long. The second intron could
be even longer as the genomic scaffold encoding EpOR34 did not have the 3‟ end of
the gene and the sequence after the second intron was obtained from the transcriptome
sequence data and the poly A+ tail obtained by 3‟RACE-PCR. The amino acid
sequence was also confirmed by homology to known B. mori ORs in GenBank.
Seven different freely available membrane prediction programs were used for
predicting the transmembrane domains of EpOR34 from its protein coding region.
Moth ORs are predicted to have 7TM domains (Benton et al., 2006), however not all
the prediction programs used here gave 7TMs. Since the exact placement of the start
and finish of the TM domains and the number of TMs predicted varied between the
different prediction programs, a consensus of the predicted domain regions was
developed and is shown in roman numerals on Figure 4.8. This consensus found
EpOR34 to have 7TMs and TMPred predicted an intracellular N-terminus, consistent
with the recent evidence that suggest insect ORs as having intracellular N-terminus
(Benton et al., 2006; Lundin et al., 2007; Smart et al., 2008; Jordan et al., 2009).

Identification of putative odorant receptors from Epiphyas postvittana 115
1 M E D K L V F E E V Y K I N M T C L R L N L S H 24
1 ATCTTTCAGTAAATTATAATGGAAGACAAATTAGTTTTTGAAGAAGTCTATAAGATCAACATGACCTGCCTCCGATTAAACCTCTCACAC 90
25 P S V P R D L K W L L T F I L M Q G L Y T A F N A I C L Y N 54
91 CCTTCTGTGCCGAGGGATCTCAAATGGCTCTTGACTTTTATCTTAATGCAGGGACTGTATACCGCATTCAATGCGATCTGTCTCTACAAC 180
55 L I F I N V K E N D F P N A C S N G V Y M V I Y F V V T F K 84
181 TTGATATTTATCAACGTAAAAGAAAATGATTTTCCCAATGCATGCAGCAACGGTGTTTATATGGTTATTTACTTTGTAGTAACATTTAAA 270
85 Y G V M V W Y Q K D I K D V I R Y Q Q E Y F D S F R E Y T V 114
271 TATGGTGTGATGGTGTGGTATCAGAAGGATATAAAGGATGTTATACGATATCAGCAGGAATACTTTGATTCTTTCCGAGAATATACTGTT 360
115 E E Q A V V K D Y I Q R G Q W V S K L W L R S T I V T A G M 144
361 GAAGAGCAAGCTGTAGTAAAAGATTACATCCAAAGAGGGCAGTGGGTATCGAAGTTATGGCTTAGATCCACTATCGTCACTGCGGGTATG 450
145 F P V K S F I D S A Y S A Y A G D F R L H S F N E N S Y G P 174
451 TTTCCTGTTAAGAGTTTCATTGATAGCGCTTATTCAGCCTACGCTGGAGATTTCAGGCTACATAGCTTCAATGAGAACAGTTATGGTCCG 540
175 Y I D E I K G R V D V F I L M Y A I F S V Y T T Y T A I M Y 204
541 TATATTGACGAAATCAAGGGCAGGGTAGATGTATTCATTTTAATGTACGCCATATTCAGTGTGTATACTACTTACACCGCAATCATGTAT 630
205 S G F A P F G P L C I L N A C A Q M D I V M M R V N H L F D 234
631 TCGGGCTTTGCGCCTTTTGGGCCACTTTGTATACTGAACGCCTGTGCTCAGATGGACATAGTAATGATGAGAGTCAATCACCTTTTCGAC 720
235 E G F D K E K S P K Q L Q N L V K F T Q N I Y G ▼ F V D Q I N 264
721 GAAGGTTTCGACAAGGAAAAATCACCGAAGCAGTTACAAAACTTAGTAAAGTTTACACAAAATATATACGGGTTTGTTGATCAAATTAAT 810
265 D I F Q V L Y E M C L K A S A I L I P I S L Y L I I E ▼ G F S 294
811 GATATATTTCAAGTTTTGTACGAGATGTGTTTAAAAGCCTCTGCGATATTAATACCTATTTCACTGTATTTGATAATTGAGGGTTTCAGT 900
295 E G K L Y Y D Y V M F S Y M A S L L C F V P C Y Y S D Y L K 324
901 GAGGGCAAGCTGTACTATGACTACGTGATGTTTTCTTACATGGCGTCTTTACTCTGCTTTGTGCCTTGCTATTATAGCGATTATCTCAAG 990
325 E K G D D L R C A I Y A S G W E K F Y D R N T R V T L R I M 354
991 GAAAAGGGTGACGATCTGCGTTGCGCAATATACGCGTCGGGCTGGGAGAAATTCTACGACAGAAATACCAGAGTCACCCTCCGCATAATG 1080
355 L I R A T R S L S I K T V F R A V C L E A F S D L C K E A Y 384
1081 CTGATACGAGCGACGAGGTCCCTCTCCATAAAGACGGTGTTCAGGGCTGTCTGCCTGGAAGCCTTCTCTGATTTGTGTAAAGAAGCTTAC 1170
385 V I F N M M Y A V L H 396
1171 GTTATTTTCAATATGATGTATGCTGTTTTGCATTAAGTCAGCTTGATGATTTAAATTTTATTCGGGCACAAACGGTACAATACTCATACT 1260
1261 TATAATTGCACAATTTAGTTTGGGTCACCTTGAGAGCTAAAAGCTAAAGAATCCTTTCAGCGGAATCCAACAAACTTTACTTGTACTAAT 1350
1351 TATCCACACACTTTGCAAATTTTATTGTTGTCATAATTCAAGTTCTTCACATATTTTAATAAACTCTTTCACACCACAAAAAAAAAA 1437
Figure 4.7: EpOR34 cDNA nucleotide sequence showing the predicted start
methionine in green, the theoretical protein coding region above the nucleotide
sequence and the stop codon at the end of the coding region as well as upstream of
the translation initiation site in purple. The predicted polyadenylation site and the poly
A+ tail are shown in red.

Identification of putative odorant receptors from Epiphyas postvittana 116
TMMOD
DAS
TMPRED
SPLIT
TOPPRED
HMMTOP
TMHMM
N L1 L2 L3 L4 L5 L6
C
Intracellular
I II III IV V VI VII
0 50 100 150 200 250 300 350
400
Figure 4.8: Schematic representation of the consensus of the predicted TM regions
from seven TM prediction programs. The black blocks represent the predicted TM
regions by the respective program on the left, and are drawn to scale, corresponding to
the predicted coding region of the gene, as indicated by the ruler at the bottom.
Number I to VII are the seven consensus TM domain regions, N- and C- terminus are
indicated at the ends and the intercellular loops indicated by L1 to L6.
The transmembrane prediction program, TMHMM2.0 predicted five TM domains
(TM1, 2, 4, 5 and 6) for EpOR34 with high probability, TM3 with low probability
while TM 7 was not predicted at all. These results were compared with the TM
prediction from TMMOD program. This predicted TM 1-6 with high probability
Residue
while TM7 was also predicted albeit with lower probability.

Identification of putative odorant receptors from Epiphyas postvittana 117
I II III IV V VI VII
Figure 4.9: Transmembrane domain prediction of EpOR34 with TMHMM 2.0
algorithms. The y-axis shows the probability of an amino acid to be part of a TM
domain. The numbered red shaded areas indicate predicted TM domains.
I II III IV V VI VII
Figure 4.10: Transmembrane domain prediction of EpOR34 with TMMOD
algorithms. The y-axis shows the probability of an amino acid to be part of a TM
domain. The numbered red shaded areas indicate predicted TM domains.

Identification of putative odorant receptors from Epiphyas postvittana 118
These results were then compared with TM predictions using five other programs (as
stated in section 4.2.16). Not all of these programs predicted the classical topology of
7TMs for ORs. This could in part be due to the lack of structural information in the
protein database for closely related proteins. Therefore, a consensus of the output
from the different prediction programs was taken in order to assign TM domains to
EpOR34. The placement of these TM domains correlate with the TM domains
predicted for EpOR1, 2 and 3 in Jordan et al. (2009), and are shown in numerals on
Figure 4.4.
To draw a topology map of EpOR34, the TM predictions produced by the consensus
(numbers I to VII on Figure 4.8) was used. A short C-terminus was predicted by both
SPLIT and HMMTOP, comparable to the short C-terminus predicted for the
topologies of EpOR1, EpOR2 and EpOR3, perhaps a feature conserved in insects
(Jordon, 2006). Some Drosophila ORs are also predicted to have short C-termini
(Clyne et al., 1999; Gao and Chess, 1999; Dobritsa et al., 2003; Kiely, 2008). This
could just be an artefact of the prediction algorithms used and only the solved
structure of such a protein will be able to confirm this theoretical observation.
N
Figure 4.11: Theoretical membrane topology of EpOR34 from the consensus of the
predictions of seven different TM prediction algorithms. N = N- terminus and C = C-
terminus.
C

Identification of putative odorant receptors from Epiphyas postvittana 119
4.4 Discussion
A total of 52 ORs have so far been identified using three different methods. Sanger
sequencing identified three ORs, 454 transcriptome sequencing identified 29 ORs and
47 ORs were identified from low coverage whole genome sequencing of E.
postvittana. Some ORs were identified by all three sequencing methods (three ORs),
some by two (21 ORs) and some by only one method (28 ORs), as summarised in
Table 4.6. Sanger sequencing gives longer sequence fragments hence contig assembly
is easier and results in long contigs being assembled; however the drawback of this
sequencing technology is the low depth of coverage, with only three ORs being
identified from Sanger sequencing of E. postvittana antennal cDNA (Jordan et al.,
2009). ORs are lowly expressed genes and due to their low copy number in cDNA
populations are not efficiently picked up in shallow Sanger sequencing. 454
transcriptomics was more efficient at identifying E. postvittana ORs, as it was able to
identify the three ORs identified by Sanger sequencing and a further 26 new ORs. The
drawback of this system was the short read lengths obtained hence the OR sequences
were in small fragments. Low coverage whole genome sequencing was able to
identify 47 ORs. This is consistent with recovery of ORs from other moths such as B.
mori where whole genome sequencing recovered most of the ORs. However, due to
the presence of introns in insect ORs, prediction of coding regions of genes becomes a
challenge.
The degenerate PCR approach taken to identify new putative ORs from E. postvittana
did not yield any significant results. This may be attributed to the low sequence
homology between insect ORs, as only a few ORs have been identified using this
method thus far (Mitsuno et al., 2008; Patch et al., 2009). Regions of homology
among ORs are limited and result in high levels of degeneracy in designed primers.
This leads to non-specific binding and amplification of undesirable genes, or genes
present in high copy numbers in cells.
In the Lepidoptera B. mori, 68 ORs (from both adult and larvae) have been identified
so far, which is higher than the number of ORs identified in E. postvittana to date.
Both these lists may be incomplete and further ORs might be identified from both
these moths, however, for the purpose of this discussion, the 68 ORs identified in B.

Identification of putative odorant receptors from Epiphyas postvittana 120
mori (Wanner et al., 2007; Tanaka et al., 2009) and the 52 ORs identified in Jordan et
al. (2009) and in this study from E. postvittana will be looked at. From pairwise
sequence alignment of E. postvittana ORs with B. mori ORs (Table 4.6), 24 one-to-
one orthologs of E. postvittana and B. mori ORs were identified, with pairwise amino
acid identity ranging from 15% to 88%. The lowest pairwise amino acid identity is
shared between EpOR9 and BmOR4 while the highest homology is shared by EpOR2
and BmOR2. Eleven EpORs share more than 50% amino acid identity with their B.
mori homologues suggesting conservation of some ORs to a certain extend within this
highly divergent group of proteins. Members of the highly conserved OR2 clade have
been shown to be co-expressed with other ORs in nearly all ORNs and may function
in transport of the ORs to the dendritic membrane (Larsson et al., 2004; Neuhaus et
al., 2004; Nakagawa et al., 2005). A similar function of EpOR2 is predicted based on
its high homology to other members of this clade. EpOR3 has previously been shown
to be highly conserved in some moth species and share 65% amino acid identity with
its B. mori homologue, BmOR49J (Jordan et al., 2009). Based on the high levels of
shared amino acid identity and functional conservation of EpOR2 and EpOR3 with
their B. mori counterparts, it was hypothesised that more EpORs will share these high
conservation levels with the B. mori ORs. Indeed nine more ORs have been identified
in the E. postvittana genome (namely EpOR4, 8, 13, 17, 27, 29, 38, 39 and 47) that
share medium to high levels of amino acid identity (between 52% to 71%) with their
B. mori counterparts, suggesting evolution of these ORs occurred before divergence
of the Tortricidae from the Bombycidae. EpOR8 shares 71% amino acid identity with
BmOR8, which is a larval-specific OR in B. mori. Based on this high level of amino
acid identity, it can be postulated that EpOR8 is a larval-specific OR in E. postvittana.
Interestingly, EpOR3 and BmOR49J (65% shared amino acid identity) bind citral, an
oviposition deterrent of E. postvittana (Jordan et al., 2009). If shared amino acid
identity conferred functional conservation on ORs, then EpOR4 (shares 60% amino
acid identity with BmOR56) would bind cis-jasmone, the ligand of BmOR56 and
EpOR29 (shares 57% amino acid identity with BmOR29) would bind linalool, citral
and linalyl acetate, the ligands of BmOR29 (Tanaka et al., 2009). However, this is
only a postulation and functional analysis of these E. postvittana ORs would confirm
their ligands.
Expansions can also be seen to have occurred in the E. postvittana OR repertoire, with
13 B. mori ORs having 28 homologues in E. postvittana. BmORs1, 4, 13, 14, 15, 16,

Identification of putative odorant receptors from Epiphyas postvittana 121
27, 28, 29, 49 and 68 have two homologues, while BmORs 26 and 30 have three
homologues in E. postvittana OR repertoire, suggesting the E. postvittana ORs within
these clades duplicated after diverging from their B. mori homologues.
Sexually dimorphic expression of ORs have been documented in some Lepidoptera
species and is one of the criteria used for identifying species-specific sex pheromone
receptors. To date sex-specific pheromone receptors have been identified from B.
mori, H. virescens, H. armigera, H. assulta, M. sexta, M. separate, D. indica and P.
xylostella; some based on tissue expression analysis (qRT-PCR and in situ
hybridisation experiments) and some based on their phylogenetic relatedness to PRs
in other moths that bind pheromone components in functional characterisation assays
(Krieger et al., 2004; Sakurai et al., 2004; Krieger et al., 2005; Mitsuno et al., 2008;
Patch et al., 2009; Große-Wilde et al., 2010; Zhang et al., 2010). Most male-specific
moth PRs cluster together on the same phylogenetic lineage based on amino acid
identity. BmOR1, BmOR3, DiOR1, MsOR1, PxOR1 and HvOR13 have been shown
to be expressed highly in male moth antennae and bind female released sex
pheromone components. Two EpORs, EpOR1 and EpOR6 also belong to this PR
clade, with both sharing 17-38% amino acid identity with BmOR1. EpOR1 is
expressed at similar levels in both male and female antennae and has been shown to
be the receptor for plant volatiles in Chapter 2, suggesting that even though it shares
the highest amino acid identity with PRs from other moths, it is not the PR of E.
postvittana. This is supported by other non-pheromone receptor members of this clade
such as BmOR9 being expressed in similar levels in both male and female antennae
(Krieger et al., 2002; Wanner et al., 2007) and BmOR4, BmOR5, and BmOR7 are
expressed in both male and female antennae albeit at higher levels in male antennae
(Wanner et al., 2007). Functional characterisation of BmOR4 and BmOR5 against the
sex pheromones of B. mori have not yielded any ligands for these two ORs yet
(Nakagawa et al., 2005), indicating that perhaps these are receptors for plant volatiles
or for unidentified pheromone components, providing support for EpOR1 being an
OR for plant volatiles and rendering EpOR6 as a possible PR of E. postvittana. If the
E. postvittana PR is a member of the PR clade, then based on amino acid identity,
EpOR6 can be postulated to be a PR candidate in E. postvittana. However, tissue
expression analysis and functional characterisation of EpOR6 is required to resolve its
role in E. postvittana olfaction.

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).

Identification of putative odorant receptors from Epiphyas postvittana 124
Of the four potential candidate PRs identified from E. postvittana based on amino
acid identity and sex-biased expression, the full length cDNA was obtained for only
EpOR34. Seven TM domains are predicted for EpOR34 by a consensus using seven
different prediction programs, comparable to the predicted TM domains obtained for
EpOR1, 2 and 3; and keeping with the notion of seven TM domains for moth ORs. An
alignment of the predicted TM domains of these four E. postvittana ORs show that the
TM regions are conserved across the four ORs. Furthermore the general locations of
the predicted TMs of the E. postvittana ORs align with the predicted TM domain
regions of the H. virescens ORs (Krieger et al., 2002) and a consensus of the B. mori
homologues. This indicates that the relative positions of the TM domains in ORs may
be conserved across the Lepidoptera. A short C-terminus was predicted by both
SPLIT and HMMTOP, comparable to the short C-terminus predicted in topologies of
EpOR1, EpOR2 and EpOR3, perhaps a feature conserved in insects (Jordon, 2006).
Some Drosophila ORs have also been shown to have short C-termini (Clyne et al.,
1999; Gao and Chess, 1999; Dobritsa et al., 2003; Kiely, 2008). This could just be an
artefact of the prediction algorithms used and only the solved structure of such a
protein will be able to confirm this theoretical observation.
Finally, the stage has been set for identification and characterisation of the PRs of E.
postvittana, with four candidates PRs being identified. Further work into the tissue
localisation through in situ hybridisation experiments and functional characterisation
either using in vivo or in vitro characterisation assays will confirm the roles of these
receptors in E. postvittana olfaction.

5.1 Introduction
5
Concluding Discussion
Moths of the order Lepidoptera have implications as pests of the agricultural and
horticultural industries of countries the world over. Feeding by larvae on and
damaging leaves and fruits of important agricultural crops have vast economic impact.
These pests have thus far been dealt with using chemical sprays, biological controls
and mating disruption, however, the never ending resistance to these agents and
impact of insecticides on human health call for the development of improved,
sustainable control measures. Studies on insect reproduction and host selection have
shed light on the involvement of the olfactory system in the survival and reproduction
of insects. For example, mate attraction studies have shown pheromones can be used
for pest control (Suckling and Karg, 1999). Olfaction has thus become the forefront in
the study of insect pests for development of new control strategies. The mechanisms
involved in odorant reception at the molecular level are still not fully understood,
however, individual proteins involved in this system are beginning to be identified
and their specific roles deduced. A better understanding of the functioning of the
olfactory system as a whole will lead to sustained pest control. The light brown apple
moth is a very important agricultural pest of the New Zealand economy as a zero
tolerance of egg, larvae, pupae and adult moth is required for all exports. With an
agricultural based economy, appropriate control measures have to be in place to meet
this stringent control measure.

Concluding Discussion 126
The aims of this thesis were to decode the binding partners of EpOR1, a member of a
group of receptors shown to be essential in odorant perception in insects; to show the
role, if any, of E. postvittana GOBP2 in odorant binding by EpOR1 in a heterologous
expression system; and to expand the known OR repertoire of E. postvittana and
identify potential candidates for the PR(s) amongst these.
5.2 Summary of results and discussion
5.2.1 EpOR1 characterisation
EpOR1 was previously identified from a male antennal EST library of E. postvittana
by Jordan et al. (2009). EpOR1 forms a clade with ORs from other moths, including
PRs from B. mori, H. virescens, P. xylostella, D. indica and M. separate (Krieger et
al., 2002; Krieger et al., 2004; Krieger et al., 2005; Nakagawa et al., 2005; Mitsuno et
al., 2008). However qRT-PCR analysis did not show male-biased expression for
EpOR1 so a functional analysis of EpOR1 was conducted to identify its binding
repertoire. Transient expression of EpOR1 in insect Sf9 cells revealed that EpOR1
binds a range of plant volatiles in vitro (Table 2.1). Some of these volatiles have
implications in plant based insect repellents (geraniol), act as oviposition deterrents
(citral), are released by plants under herbivore attack (methyl salicylate) and are
natural constituents of plant aroma (geranyl acetate). This characterisation of EpOR1
reveals it to be a receptor for general odorants and shows that at least some moth ORs,
like the Drosophila ORs are broadly tuned, recognising a range of compounds. The
decoding of binding partners of ORs has implications in dealing with insect pests by
targeting their chemosensory systems. This will aid to the development of sustained
and environment friendly insecticides.
5.2.2 EpGOBP2 reconstitution of the Sf9 cell assay
E. postvittana GOBP2, identified by Newcomb et al. (2002) was successfully used for
reconstituting the Sf9 cell assay system for functional characterisation of EpOR1.
Recombinant EpGOBP2 was able to bind seven of the ten compounds tested in a

Concluding Discussion 127
VOBA setting (Figure 3.5). These results indicate that EpGOBP2 is broadly tuned
recognising a range of compounds. EpGOBP2 was further tested for its ability to
solubilise two ligands of EpOR1, methyl salicylate and geranyl acetate in the Sf9 cell
assay. These experiments showed that EpGOBP2 could be used to replace DMSO as a
solubilisation agent for both methyl salicylate and geranyl acetate (Figures 3.7 and
3.8). EpGOBP2 also enhanced the sensitivity with which EpOR1 recognise these
compounds in vitro as determined by comparing EC50 values (Table 3.1). The
increased sensitivity observed for EpOR1 in the presence of EpGOBP2 could be a
combination of a role as a solubilising agent and/or as an activated ligand and suggest
that species-specific OBPs can be used to reconstitute the Sf9 cell assay system for
improving the sensitivity of the system. This is the first documented role of a
lepidopteran GOBP in odorant binding in an in vitro OR characterisation assay.
5.2.3 Identification of putative ORs from E. postvittana
The initial development of an antennal EST library at Plant and Food Research Ltd
enabled the identification of three ORs from E. postvittana (Jordan et al., 2009). A
further 49 ORs have been identified as part of this project using 454 transcriptome
and Illumina genome sequencing (Table 4.6). These ORs share some common
features with B. mori ORs identified so far. Twenty-four E. postvittana ORs have one-
to-one orthologs with B. mori ORs. Expansions seem to have occurred in 13 of the B.
mori ORs, with E. postvittana having between 2 to 4 ORs for each of the 13 B. mori
ORs. EpOR1 and EpOR6 fall into the moth PR clade. However, EpOR1 has been
shown to be the receptor for plant volatiles, with similar expression levels in male and
female antennae. For EpOR6 tissue expression analysis by qRT-PCR has yet to be
performed. The other possibility is that some of the phylogenetics is not correct as not
all ORs are full length and members of the PR clade may be placed elsewhere
currently. Only the full lengths of all the identified E. postvittana ORs will determine
their true phylogeny. It could also be that the PRs are missing from the list of
identified ORs, as the E. postvittana OR repertoire is approximately twenty ORs short
compared to B. mori OR repertoire, thereby indicating that not all E. postvittana ORs
have been identified. Since the PRs from six other moth species fall into the moth PR
clade, we assume the E. postvittana PR(s) to be part of this clade also. On the other
hand, not all members of this clade are PRs (for example BmOR4, 5, 7 and 9, as

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

Concluding Discussion 130
components or test odorants may be toxic to the OR being tested and an
understanding of the roles of other olfactory players in these in vitro systems may
help overcome these limitations.
Functional studies of EpOR6, 30, 33 and 34 will enable the determination of their
roles in E. postvittana olfaction. The phylogenetic relatedness of EpOR6 to the
pheromone receptors from other moths confers it as a PR candidate, however, tissue
expression analysis and functional characterisation of this candidate OR will deduce
its role in E. postvittana olfaction. The relatively high expression of EpOR30, 33 and
34 in male moth antennae, as shown by qRT-PCR is suggestive of a plausible role in
pheromone recognition of these ORs. In situ hybridisation of these candidates can be
used to test if they are likely PRs. ORs that are expressed at the base of pheromone
sensitive trichoid sensilla of male moths will likely be PRs. Functional analysis by
cloning, expression and characterisation in an assay system against E. postvittana
pheromone components will confirm their binding partners. The functional
characterisation of PRs from moths such as B. mori and H. virescens has indicated the
presence of multiple PRs within each species which are tuned to recognising one or
more of the sex pheromone components. It is tempting to postulate the existence of
multiple PRs in E. postvittana also and one or more of the four candidates could be
the PRs of E. postvittana, however, further functional analysis will have to be done to
test the roles of these ORs.
If any of these four candidates are found to be the PR for E. postvittana, then further
strategies can be developed in dealing with this agricultural pest. Mimics that can act
as irreversible blocking agents for the binding site of the PR could be developed. A
mimic that will not be cleared from the receptor after signal attenuation would be
ideal (native pheromones are cleared from the receptor hence large amounts are
needed in mating disruption studies, rendering this pest control method expensive). Its
release into E. postvittana infested areas could see a reduction in the number of
successful matings as the mimic will flood the PR and confuse the male moth.
Another strategy would be to genetically modify the PR itself so that it no longer
recognises the pheromone components. This can be done by completely shutting off
the PR by RNAi silencing. Small interfering RNA or double-stranded RNA (dsRNA)
could be sprayed on host plants, or genetically modified plants expressing dsRNA

Concluding Discussion 131
against the receptor could be introduced (pertaining ethical approvals). Larvae feeding
on these plants will have their PRs turned off or modified, hence will be unable to
recognise sex pheromones and the number of successful matings will decrease. Turner
et al. (2006) fed dsRNA to larvae and demonstrated the effective knockdown of genes
expressed in the adult antennae (Turner et al., 2006). This method will however
require the GM plants, siRNA and dsRNA sprays to undergo various tests to render
them safe for the environment and humans.
During the course of this project, 49 putative ORs were identified, however, the full
length sequences of 48 of these still need to be completed. Towards the end of this
study, six more lanes of Solexa genome sequences was obtained. However the data
was not available on time to include in this thesis. This new data combined with the
previous genomic sequence dataset will hopefully give longer scaffolds with longer, if
not full length coding regions of the putative ORs. Further novel ORs could also be
identified from the new data, thereby increasing the receptor repertoire of E.
postvittana. Functional characterisation of these full length ORs can be done to
identify male and female specific ORs and their binding components. A detailed
functional characterisation of these ORs will facilitate the decoding of all these ORs,
and an odorant–OR map can be built for E. postvittana. Such a study will provide data
that can be used as a basis to create a computer model to predict binding partners for
ORs in related moths. A computer model that can for example narrow down on „x‟
number of candidate odorants for testing an OR with can be used as a first screening
towards characterising novel ORs. This will save the researcher physically testing tens
to hundreds of compounds per receptor to identify its binding components.
The only lepidopteran for which a draft genome sequence is publically available to
date is the silkmoth B. mori. This is a domesticated moth which is dependent on
humans for survival and reproduction. Currently it is used as the model for
Lepidoptera studies, including tortricid moths however, the availability of genome
data for an insect pest will be invaluable to Tortricidae genomic biology, especially in
understanding the underlying molecular mechanisms of insect pests and combating
the disruptive members of this family. Keeping this in mind, the genome sequencing
of E. postvittana has been initiated. E. postvittana is perhaps one of the most
disruptive members of this family, in that it is highly polyphagous and can survive in

Concluding Discussion 132
a wide range of temperatures. The availability of a draft genome of E. postvittana will
be the first genome for a pest member of this family and will become the model
organism for tortricid pests. The availability of the genome sequence of E. postvittana
should enable functional genomic studies of the moth; provide a wealth of
information on genes involved in biological processes and in the regulation of gene
expression as well as facilitate the study of the complexity of gene families. Studies of
host-plant specialisation for example, E. postvittana is polyphagous so genes involved
in its survival on such a wide host range, as well as proteins involved in food
digestion in the gut of the larvae can be identified. Other target processes include the
regulation of genes involved in the survival of E. postvittana, such as for juvenile
hormone, which have implications in physiological processes of moths can also be
studied in detail. Other genes associated with the olfactory system and their regulation
mechanisms could also be studied in detail. One of the major drawbacks of using
chemical insecticides is development of resistance by the insects. These resistance
mechanisms can also be studied and better insecticide targets developed. With the
availability of genomic data for more moths, the evolution of ORs can be studied. An
improved set of ORs from lepidopterans can perhaps be used in phylogenetic studies
to test for associations, if any, between phylogenetic relatedness and odorant binding.
The problem with any ab initio whole genome assembly is the feasibility of the
assembly process. E. postvittana does not have any closely related genome sequence
available for mapping its genome to. The low cost of sequencing with high throughput
sequencing platforms has the drawback of producing significantly short reads that are
impossible to assemble ab initio. Another problem facing E. postvittana genome
assembly is that its genome size is unknown. B. mori genome has been shown to have
large repeats and it is highly likely to be the case for E. postvittana. Assembling such
a genome will be highly challenging.
Fortunately for the genome sequencing project, a bacterial artificial chromosome
(BAC) library of E. postvittana has already been constructed. BAC-end sequencing
will enable local, smaller assemblies of individual BACs which can be used further to
assemble longer scaffolds, thereby facilitating the assembly of the draft genome.
Several BACs that potentially have genes for several of the putative ORs have already
been identified. These BACs can be used as the starting point for BAC shotgun

Concluding Discussion 133
sequencing and assembly. The genome sequence dataset that we currently have has
lots of gaps, as shown by the high number of scaffolds, approximately 843000
scaffolds. This coverage is being improved by further sequencing of the genome.
Another complementary approach to BAC sequencing is mate pair end sequencing
(Illumina, 2010), which will again give the ends of 5kb long fragments that can be
used as a guide for assembling the shotgun sequencing fragments.
The challenges faced within the context of this thesis in identifying new ORs from E.
postvittana is reflected by the low levels of homology between the members of this
group and the importance of high throughput sequencing technologies to such
projects. With the identification and decoding of increasing number of moth ORs,
important questions about correlation of the phylogenetic relatedness of these proteins
with their ligands, as well as the occurrence and significance of multiple PRs in moths
can be addressed.

Appendix A 134
A.
Appendix A – Dose
response profile of pIB-
V5 His empty vector
control to methyl
salicylate
Figure A.1: Dose response of the empty pIB-V5 His vector to methyl salicylate in the
concentration range of 10 -5 M to 10 -13 M. Error bars represent the standard error of 6
responding cells.

Appendix B 135
Buffered TB (Sambrook et al., 1989)
B.
Appendix B –
EpGOBP2 expression
buffer recipe and
protein details
Per Litre: K phosphate
900mL water Per Litre:
12g Tryptone 23.1g KH2PO4
24g yeast Extract 125.4g K2HPO4
4mL glycerol Autoclave
The TB was autoclaved and cooled to room temperature. 100 mL of sterile K
phosphates was then added to it.
His6-EpGOBP2 has a molecular weight of 22597.20 Daltons and the amino acid
sequence is as follows:
M H H H H H H S S G L V P R G S G M K E T A A A K F E R Q H M D S P D L G
T D D D D K A M A D I G S E F E N L Y F Q T A E V M S H V T A H F G K A L
E Q C R E E S G L S T A V L E E F Q H F W R D D F E V V H R E L G C A I L C
M S N K F S L M Q D D A R M H H E N M H D Y V K S F P Q G E V L S A K M
V E L I H N C E K P Y D D I K D D C E R V V K V A A C F K V D A K K A G I A
P E V A M I E A V M E K Y
Native EpGOBP2 has a molecular weight of 16094.51 Daltons and the amino acid
sequence is as follows:
TAEVMSHVTAHFGKALEQCREESGLSTAVLEEFQHFWRDDFEVVHRELGCAI
LCMSNKFSLMQDDARMHHENMHDYVKSFPQGEVLSAKMVELIHNCEKPYD
DIKDDCERVVKVAACFKVDAKKAGIAPEVAMIEAVMEKY

Appendix B 136
Figure B.1: Chromatogram showing the elution profile of His6-EpGOBP2 from
HiTrap chelating HP column. The numbers in red indicate the elution fractions and
His6-EpGOBP2 eluted as a single peak in fractions 16–21.
Figure B.2: Elution profile of His6-EpGOBP2 on Q-sepharose HP column. The
numbers in red indicate the elution fractions and His6-EpGOBP2 eluted in a single
peak in fractions 25–31.

Appendix C 137
C.
Appendix C – ClustalX multiple
sequence alignment of moth PR
clade
MsOR1 ------MIFMDDPLSKSIKDPRDYRYMKLFRSTLRLIGSWPGR------DLKEEGATKYE
BmOR-3 ------MIFVDDAVIG-IKDPREYRHLRVLRTSLRLLGAWPGH------YLGEETGSKYE
HR6 -MNLRKFLFENEAVEG-INSPADYLYTRILRFNLDFIRTWPRK------ELGEPENLAFT
HR16 -MGLRQFLFENEAVEG-INTASDYLYIKILRFTLVIVNSWPRK------EIGEPESPRLS
HR14 MTGIRDFFFNYEAKDG-VTNPTEYPYMIMSRHLLTVITCWPKKPKEGLNARAKLRAKIWV
HR15 MTGFRDFVFNYQPKDG-ITNPVDYPYLIIARYLLTFISMWPKKSVVYHSARAELKARIWL
HR11 ------MHLAGNAVTG-ITGPMDYKYMKVLRFVLRIISGWPGK------ALGEKTLRIEG
BmOR-5 ----MLLYYPNTQVKEKVNNVEEFTYIKFLKSFCKIMDFWP--------EREEKNSKTRI
BmOR-7 ----MLLYHPNTQVEEKVNNVEEFTYMKFLKSFCKIMDFWP--------EREEKNSKTRI
HR13 ----MKILSDGSDLEG-VEKVEDIFYINLARKSMWILDSWP--------KAFNASSKYRY
BmOR-4 MFKIIKNIIVENDALKQVEKPQEFQYMKWVQYHLKYIDGWPNM------DMNKKNVSKIR
BmOR-1 ----MLLSFKDDSRSPDIQKPQNFQYMKILRFNLKIICAWPE-------KQLNEIRSLGH
EposOR1 ------------------MDVFNLKYMRMIRFTLRSIGAWPSHEFED--VPATKLTLLSS
BmOR-6 -----------MKEEYYLQHPRTQLFYKVLAHVSTIESTIDLTWWG---YTFPKYVGWFY
.
MsOR1 IAPLYWVLVIKITCFVLTIIYLIENTNKLGFFEIGHVYITVFMTMITLSRSITLSLNPKY
BmOR-3 CAPMFLLMFIKIACLYLTIVYLRNNADVLGFFELGHVYLTIFMTFVTLSRGFSLTWNPNY
HR6 VFMQYFYLILNIVTVMGSTSYIVVRGSELSFIEAGLMYLIFLIGIVDTLTVVCLTFSEKF
HR16 AFAKYFYLFLTVLAAIGSIAYVAVHNRELTFLETGHMYIVVLMSLVDVSRVATLTMSTTY
HR14 IVQKIFHMSLCLLTTLGMAMYIGLHKKSMSFLELGHLYISLLMTVVIFSRITTLCLNPKY
HR15 WVQKFYHLLLCAVAFFGGVLYITLHKKSMTFYELGHLYISLLMMACTFSRITTLCFNDEY
HR11 MGHAYYNTILSLVYLALGIAYLKKNFHRFDFLELGQLYIVLLMNMLSTSRAFTLCLSQKY
BmOR-5 FRLRYILVLQFCFTLVAGVLYLTNSVGKQTFYDLGHTIITVLMNVVSLSRLILR-CFKKY
BmOR-7 FRLRYILVLQFCFTLVAGVLYLKNNFGKKTFYDLGHTIITVVMNVVSVSRLILR-CFKKY
HR13 F----VLALN-VATLIGGAIYLRNNTGVLSSFELGHTYITVFMNCITCSRCLMI-LSKDY
BmOR-4 FHKRHLLVVEQTITFLSQMFYIVKNYGKLSFFEIGHSYITALMTIVIFSRSVVT-ALGRY
BmOR-1 SIHRVILPIQSVVCLACGILYIHFHFNEIPFFILASTFITVMMNLVTCSRTALVMLFERY
EposOR1 YSYTCFLCIICCFGTIAQIAYLITNQGILGFIDLGQTYLTVLMCFVYIQRTMLP-LQTSY
BmOR-6 HLQCNVVRLFGKCVVVSQILFIILNYQTIDKSVFIIAITITPLGALVGIKAESA-KAECY
:: : :
MsOR1 RRVMTKYITKMHLFYYK---DMSDIALKTHIRVHKLSHFFTMYLSTQVVLGTVTFNIVPM
BmOR-3 HKVVKKFITEMHLLYFK---DNSEYAMKTHRRVHKISHFYTVFLKVQMIAGLTLFNVIPM
HR6 RVLAKDFLTKTHLFYYK---DRSKHAMEIHKKIHLISHLFSLWILFQMLSGLSLFNIIPM
HR16 REVARDFLTKIHLFYYK---DRSKHAMETHRAVHKISHLFTLWLVGQMLSGLSLFNLIPM
HR14 RAVSTEFLTKIHLFYYK---DDSEFSMQIHKQVHKISHLFTLYLTGQMIAGLSLFNLTPM
HR15 RVVAKDFVTKIHLFFYK---NRSDYSMQIHKKVHMISHVFTLYLSGQMMLGLFLFNVTPM
HR11 REVAKIFIQKIHLFYFK---EKSDYAMKIHVIVHKISFISAVYLSVLLFIAAVMFNLIPM
BmOR-5 DVVGQQFINKIHLYHYR---NDSEYAMKIHTVVHKISHNMTYIFSFCIIFGTVTFNLTPI
BmOR-7 DVVGQQFINKIHLYHFR---NDSEYSMKTYKAVHKISNNMTYIFSFSIFVCVVTFNLNPV
HR13 NHVMTLFVQKIHLFHHK---HKSDYAYLTHIFIHKISHFYTVYLLGLALNGLFLFNMIPF
BmOR-4 RKIARYFVSSLHLYHYK---DISEYALQTHLLVHRLSHYYTVYLISLVVTGMLLFNITPL
BmOR-1 LVLTGRFITVMHLFNFQ---KNSDYAYKLCTFVNRMSHFYTLYVLFSMFMGLGLFNLLPL
EposOR1 QAEIIEFSTKFHLMYHK---NETEFAAKMHNKVKRICEIVTGIQHLQIYYVLVMYNIAPL
BmOR-6 VNLMKNFMDKVHIHSIYRKNENNEFVKKKVIQIERVSRFTAYFLVILIAINCLSWMLKPT
: *: . .. :. :. : : : *
MsOR1 YNNYKVGRFEN-NILVNDSYELSIYFKTPTKFLSTLNGYIAITTFNWYSSYICSNFFCMF
BmOR-3 YNNYRQGNYAS-DRPANITYDLSIYYET-FDILNTPNGYIFICVFNWFASYICCSFFCSF
HR6 YSNLAAGKYRK-GGLQNSTFEHSLYYLYPFNTSTDITGYIIACILHWIISYLCSCWFCII
HR16 YSNYAAGRYSG-DVSKNSTFEHSLYYSYPFDTSTDIRGYSIACVIHWVLSYLCSTWFCMF
HR14 YNNFSAGKYKK-GGLKNSTFEHSLYYSYPFNASSDVGGYIVSNICDWIISYLCSTWFCTL
HR15 YNNYSAGKYKS-GGLKNSTYEHSLYFSWPFNASTDMRGYIVSNILNWMLSYTCSSWFCVI
HR11 YNNYSAGRYSSFDNLENTTYEQAISCLYPWNFETNFNGYLVATLSGWYGTMLCGSSVSMF
BmOR-5 FNNIGSDAYKN-PRPDNVTLQQCVYYALPFDYTGNFKWYLLVAIFNVQKTFFCTSLFILF
BmOR-7 FNNIGSGAYKN-PRPDNVTLQQCVYYALPFDYTGDFKWYMLVAIFNVQKTFFCTSLFILF
HR13 YNCYSRGMFRD-VIPANATYDHAVFYSVPFDYTTKFKGYLAMTSFNVFISYTCTSYFCVV
BmOR-4 YNNISSGVFNS-PRPENMTFQHAVYLGLPFDYTTDIKGYFVVFILNWHLSHIAASYFCTF
BmOR-1 YNNYVSGAFSD-PYGPNVTFFHSVYFAFPFDYSHNFRGYIIMALFNSYVSVTCSIGLVMF

Appendix C 138
EposOR1 YSNFKSGMLSS-EKPINGTYEHSVYYVLPFDHNNEV-WYPVVGLYNFYVSYNLGAMFSCH
BmOR-6 LHNIK---HFEEIMNKSMEFQYYIYFWTPLDYKYNLRDYIIIHTLCIYLGATAVTVIVTF
. : . * .
MsOR1 DLALSLLIFTVSGHFKILIHNLNNFPLPAVVSDSSKVLKTDEIQ----------APLYNK
BmOR-3 DLILSLMISTVSGHFRILIHNLLTFPLPEAITASKKFVDKHRCNGNRSEFVLEEAKLYSP
HR6 NLFLSLLVFNLWGHFKILISTLNEFPRPSSKSVDTQESP----------------YKYTE
HR16 DLFLSLMVFHLWGHFKILINTLNDFPRPSSKVEGAQ---------------------FSD
HR14 DLFLSIMVFHVWGHFKILLHDLDHFPRPANLTTFKLDNSN--IT--------LTSEKFSS
HR15 DFFLSLMVFHIWGHFKILLHDLDHFPRPLNKVNSVIEDS---IT--------ITNEMYSQ
HR11 DLFLCLMIFNLWGHFKILIHNLEHFPRPASEIVDAEGAERSGRI--------IGSEMYSQ
BmOR-5 ELSLSLMIICLWGHLRIFIHNLNHIPAPRNSFE------------------------YTK
BmOR-7 DLLLSMMIIHLWGHIRIFIHNLNHIPAPRNSLE------------------------YTR
HR13 DLTISLVIFHLWGHMRLLTYHLANFKKPASVLESNDNNKDEIKD-----------HSYTE
BmOR-4 DLFLSLLILHLWGHLRIILNNLKTFPKPYTNNS-----------------------MYTE
BmOR-1 DLLMCLMVMHVWGHLKILSHNLINFPRPKASHVITTPNGPTN-V-----------ETYTE
EposOR1 DLLISVYVFHIWGHLNICEHNLNNFPRPSITRNSKTVPLR-----------------YSA
BmOR-6 DIFNFIAVFHVVAHIQILKNNVKSNWSDD----------------------------FNE
:: : : : .*:.: : :.
MsOR1 TEKKDITLRLKQCIDYHREVLEFTQDISEAFGPMLFVYYLFHQVSGCLLLLECSQMDAAA
BmOR-3 AEMWQVTDRLRQCIDYHRKLVEFTGDISEAFGPMLFVYYLFHQVSGCLLLLECSQLNTAA
HR6 EELIEVAEKLKDCINYHREIKIFTNRMSDVFGPMLFIYYAFHQASGCLLLLECSQMTARA
HR16 EELVDVAARLKDCIVYHREITLFTDRMSNVFGPMLFVYYSFHQASGCLLLLECSQMTAQA
HR14 IELGQVSEKLKKCIEYHRKIVSFTDEMSEVFGPMLFVYYGFHQTSGCLLLLECSQMTVAA
HR15 TELDQVFDRLGKCIDYHREIVSFTDKMSEVFGPMLFAYYGFHQASGCLLLLECSQMTVAA
HR11 AELEQVAVLLRECIQYHMLIFDFTNNMSDAFGMALFIYYSFHQITGCLLLLECSQMTAAA
BmOR-5 EERQEVDDTLKKCIQHHTLIIGFVRIMSETYGLAVLIYYAFQQVVGCLLLLQCSQMELKT
BmOR-7 EERQEVDNTLKKCIQHHTLIIGFVRIMSETYGLAVLIYYAFQQVVGCLLLLQCSRLDLKT
HR13 EELKEVFSKLREYIQHHNLILEFSSEMSNAFGPALLAYMVFHQVSGCILLLECSQLDTKT
BmOR-4 EENQVVLLKLQECIRYHNFIISFTVMMSNVYDVVIIVYYLFHQVTGCLLLLQCSTLDWES
BmOR-1 EESKEVFARLRECIKHYGTVDDFANDMSETFGVILLVYYGFHQVSLCMLLLECSDLSTKA
EposOR1 EENKKVAAGLKEIIIHYIMIKKFVEKTSNTYSVTLCFYYGFHMVAECILLLQCSTLEVEA
BmOR-6 SEKK---GYLVSILEYHAYIIRIFGEVQSAFGLNVASNYLQNLIEDGLFLYQIMNGEKEN
* * . : :: : : ...:. : : ::* :
MsOR1 LMRYGLLTAVLFQQLIQLSVVVESVGTVTGYLKDAVYNVPWEYMDTQDRKTVCIFLMNVQ
BmOR-3 LVRYGVLTVVLYQQLIQLSVIVESVGTVTGRLKDAVYEVPWEYMDTSNRKTVAIFLMNVQ
HR6 LMRYLPLTIIMLQQLIQLSVIFELVGTESEKLKDAVYGVPWDCMDTKNRKVVMFFLMNVQ
HR16 LMRYVPLTIILTQQLIQLSVIFELVGSESDKLKHAVYGLPWECMDVKNRRVVLIFLANTQ
HR14 LVCYLPLTIMLFQQLIQLSIIFELVGSVSDKLKDAVYSLPWEAMDIKNKKTVAIFLMNVQ
HR15 LVRYLPLTIILFQQLIQMSIIFELVGSVTDKLRDAVYGLPWEAMDTKNRKTVAFFLMNVQ
HR11 LTRYLPLTIIMFGELVLLSIIFETIGTMSEKLKDAVYKVPWEYMDTKNRRTLLIFLIKVQ
BmOR-5 VTRFGFLTLVLNQQLIQISVIFELLGYMSDKLQDAVYCVPWEYMDTSHRKMVYMMFRQSQ
BmOR-7 ITRFGFLTTMVNQQLIQISVIFELLGYMNDKLQEAVYCVPWEYMDTSHRKMVYMMFRQSQ
HR13 LVRYGPLTIVIFQQLIQISVIFELLGSSNDKLIDGVYLVPWEYMDTKNRKLVFTMLRQSH
BmOR-4 LSRYGPLTLIIFQQLIQVSMIFEILGFLSDKLPNAVYSIPWEAMNVTNRKLVQVLLQKSQ
BmOR-1 MLRYGPLTLIMIQQLIQISIIFELLGSVADRIPDAVYQLPWECMDVKNRRVVYGFLRRTQ
EposOR1 LAKYGFLTVAVYQELIQLSVVFELIYAKGTSLIDAVYGLPWECMDNSSRRTVLILLQIVQ
BmOR-6 VLMYGLMIILYLGGLIFLSIVLEEIRRQNYDLCEYVYALPWEGMSLENQKIFVVFLQRTQ
: : : *: :*::.* : : . ** :**: *. :: . :: :
MsOR1 EPVHINALGLAKVGVQAMAGILKTSFSYFAFLRTVSN---
BmOR-3 EPLHVNALGLAKVGVQSMAAILKTSFSYFTFLRTVSE---
HR6 EPVHVKAMGLANVGVTTMASILKTSLSYFTFLLSQTKEE-
HR16 EPVHVKAMGVANVGVTSMAAILKTSMSYFTFLRSM-----
HR14 EPVHVKALGLAEVGVTSMTAILKTSMSYFTFLRSK-----
HR15 EPVHVKALGLAEVGVTSMTAILKTSMSYFAFLRSM-----
HR11 EPIHVKAGGLVDVGVTTMASILKTSFSYFAFLRTF-----
BmOR-5 IPLQLKAMNMLSIGVKTMVSILKTSVTYYLILKTVTTD--
BmOR-7 IPLQLKAMNMLSIGVKTMASILKTSVTYYLMLKTITANEA
HR13 RSINLTMMSMVTVGVQTMTAILKTSFSYFVMLKTVAEEE-
BmOR-4 KPIQFKAMNMMSVGVQTMASIIKTSISYFIMLRTIARD--
BmOR-1 NPVRFKAMGMLDVGVQTMASILKTSISYFVMLRTVAT---
EposOR1 QPLSLKACGMVPVGIQTMQAILKGSFSYFLMLRTFANQ--
BmOR-6 PDLEFETVCGMKAGVKPAFSIVKSMFSYYVMINSRF----
: . *: . .*:* .:*: :: :
Figure C.1: ClustalX multiple sequence alignment of the moth PR clade, including
members from B. mori, M. sexta, E. postvittana and H. virescens (Krieger et al., 2002;
Krieger et al., 2004; Nakagawa et al., 2005; Wanner et al., 2007; Jordan et al., 2009;
Patch et al., 2009). Amino acids in red indicate region of degenerate primer design for
identifying PR genes in E.postvittana.

Appendix D 139
D.
Appendix D – Solutions for
nuclear DNA isolation
Table D.1: Reagents for making 1 L of Nuclei Extraction Buffer, pH 6.
Reagent 1 L Final concentration
Mannitol 91g 0.5 M
PIPES-KOH 3.78 g 10 mM
MgCl2 7H2O 2.03 10 mM
PVP K30 20 g 2%
L-lysine monohydrochloride 36.52 g 200 mM
EGTA 2.28 g 6 mM
Sodium meta bisulphite 1.9 g 10 mM
800 mL of deionized water was heated up in the microwave for 2-4 minutes and
poured into a glass beaker with a magnetic stirrer. PVP K30 was added to the beaker
with vigorous stirring. Mannitol was added and completely dissolved before addition
of PIPES, magnesium, lysine, and EGTA. Volume was adjusted to 1 L, pH 6. The
buffer was split into two 1 L flasks, autoclaved and cooled to room temperature. The
buffer was kept at 4°C until required.
Just before starting the nuclei isolation, 0.9 g sodium meta-bisulphite was added to
each flask. To one flask 0.2 mL -mercaptoethanol was added and this flask labelled
as NEB complete buffer, while the other flask is NEB– no -mercaptoethanol. Both
the flasks were kept on ice until used.
The lysis buffer in part II of nuclear DNA isolation consists of 0.5% SDS, 5 mM
EDTA, 150 mM Tris-borate pH 7.4. To prepare this solution the Tris buffer was
titrated with boric acid, instead of the usual HCl, autoclaved and kept at room
temperature.

Appendix E 140
E.
Appendix E – Multiple
sequence alignment of E.
postvittana ORs together with
ORs from five other moth
species

Appendix E 141
I II

Appendix E 142
III IV

Appendix E 143
V

Appendix E 144
VI VII

Appendix E 145
VII continued
Figure E.1: Multiple sequence alignment in ClustalX of E. postvittana ORs with
those of five other species B. mori (Wanner et al., 2007), H. virescens (Krieger et al.,
2002, Krieger et al., 2004) and P. xylostella, M. separata, and D. indica (Mitsuno et
al., 2008). The sequences were obtained from GenBank. Transmembrane positions
obtained from consensus of EpOR1, 2, 3 and 34 are shown as orange bars above the
alignment.

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