CA2616144A1 - Insect olfactory receptors and ligands therefor - Google Patents

Abstract

The present invention relates to insect olfactory receptors, as well as functional variants and mutants thereof. The invention also relates to polynucleotides encoding these olfactory receptors, as well as vectors comprising said polynucleotides.
Furthermore, the present invention relates to methods of identifying odorant ligands, methods of identifying compounds that modulate receptor activity, as well as biosensors comprising said receptors.

Description

DEMANDES OU BREVETS VOLUMINEUX
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INSECT OLFACTORY RECEPTORS AND LIGANDS THEREFOR
FIELD OF THE INVENTION
The present invention relates to insect olfactory receptors, as well as functional variants and mutants thereof. The invention also relates to polynucleotides encoding these olfactory receptors, as well as vectors comprising said polynucleotides.
Furthermore, the present invention relates to methods of identifying odorant ligands, methods of identifying compounds that modulate receptor activity, as well as biosensors comprising said receptors.
BACKGROUND OF THE INVENTION
The detection and location of mating partners and host-plants are critical insect behaviors that depend in part on the sense of olfaction. Odorant receptors (Or) form a large and diverse family primarily responsible for the molecular recognition of olfactory stimuli in the insect's environment. In insects they are expressed on the surface of dendrites that extend into a lymph filled interior of sensory hairs or sensilla.
Buck and Axel (1991) first identified the vertebrate odorant receptors as members of a large family of G-protein coupled receptors (GPCR), but because of extreme sequence divergence, insect Ors were not discovered until the Drosophila melanogaster genome was sequenced (Clyne et al., 1999; Gao & Chess, 1999; Vosshall et al., 1999;
Robertson et al., 2003). Much progress has since been made in our understanding of the molecular and neurological mechanisms of insect olfaction, primarily in D.
melanogaster, including the principle that each sensory neuron, characterized by a single type of receptor, projects to a single glomerulus (with some notable exceptions, for example, see recent reviews on the subject by Rutzler and Zwiebel (2005) and Hallem et al., (2006). Using this information it is now possible to interfere with the olfactory system by targeting Ors (Larsson et al., 2004), ultimately to disrupt pest behaviors for the purpose of developing new insect control technologies. For example, a mosquito Or that responds to components of human sweat has been identified (Hallem et al., 2004) and efforts are underway to develop molecular approaches that alter the olfactory-mediated host seeking behaviors of insect disease vectors (http://www.gcgh.org/subcontent.aspx?SecID=389). Despite the progress being made with dipteran Ors, few odorant receptors have been identified from the Lepidoptera, a taxonomic group that includes numerous pest species causing substantial economic losses to global agriculture and forest production systems.
The silkworm Bombyx mori is one model for insect olfactory research, particularly the male moth's legendary sensitivity and selectivity for female sex pheromone (Schneider, 1992). Specialization of the male olfactory system (sexual dimorphism) contributes to the male moth's ability to detect minute amounts of the female pheromone. While several different types of sensilla are found on male silkworm antennae (Pophof, 1997), the pheromone sensitive trichoid sensilla are the most numerous, with approximately 17,000 on a single male antenna (Heinbockel and Kaissling, 1996). Each trichoid sensillum on the male antenna houses two sensory neurons tuned to one of the two main pheromone components, bombykol or bombykal (Heinbockel and Kaissling, 1996). The large number of pheromone sensitive neurons project to enlarged glomeruli (termed the macroglomerular complex) found on male (but not female) antennal lobes (reviewed in Hildebrand and Shepherd, 1997; Ai and Kanzaki, 2004).
The sexual dimorphism of male moth olfactory systems was used to identify the first putative pheromone Or genes. The genome sequence of B. mori (Mita et al., 2004; Xia et al., 2004), the only lepidopteran genome sequence publicly available to date, facilitated the discovery of six Ors that were identified by their amino acid similarity with published dipteran Or sequences. Based upon their high levels of gene expression in male compared with female antennae they were considered to be putative pheromone receptors (Sakurai et al., 2004; Krieger et al., 2005;
Nakagawa et al., 2005). Subsequently it has been demonstrated that BmOrl responds to bombykol and bombykal, while BmOr3 selectively responds to bombykal (Sakurai et al., 2004;
Nakagawa et al., 2005; Grosse-Wilde et al., 2006). Similarly, a total of 18 Heliothis virescens Ors have been identified from privately owned sequences, including five that are expressed at higher levels in male compared with female antennae (Krieger et al., 2002; 2004).
The olfactory mechanisms underlying host-plant seeking and selection by female moths have received less attention, and the female olfactory system is often considered to be adapted to detecting general olfactory stimuli. However, female silkworm antennae, like males, have large numbers of trichoid sensilla, approximately 6,000 per antennae (Heinbockel and Kaissling, 1996). One of the two sensory neurons within the most common female trichoid sensilla responds most strongly to benzoic acid (referred to as the benzoic acid cell) while the other responds to 2,6-dimethyl-5-hepten-2-ol and linalool (referred to as the terpene cell) (Priesner, 1979;
Heinbockel and Kaissling, 1996). Insects may use terpenoid odorants to detect and discriminate host-plants but the behavioral relevance of benzoic acid sensitivity in female silkworm antennae remains unknown (Heinbockel and Kaissling, 1996). In addition, olfactory neurons from the female trichoid sensilla project to two lateral large glomeruli (LLG) that are much larger in the female silkworm antennal lobe (Koontz and Schneider, 1987). The antennal lobes of Manduca sexta moths also have two large sexually dimorphic glomerui (King et al., 2000). Interestingly, the lateral large female glomerulus (latLFG) of M. sexta responds preferentially to antennal stimulation with linalool, particularly the positive enantiomer (Rospars and Hildebrand, 2000; Reisenman et al., 2004).
There is a need to increase the representation of Ors from the Lepidoptera by determining additional Ors from the silkworm genome. Furthermore, there is a need to identify Or genes that are expressed predominantly in female compared to male antennae, a step towards identifying female-specific olfactory pathways that can mediate important pest behaviors, such as host-plant seeking and selection for oviposition.
SUMMARY OF THE INVENTION
The present inventors have identified 41 new olfactory receptors.
Thus, in a first aspect the present invention provides a substantially purified and/or recombinant polypeptide comprising amino acids having a sequence as provided in any one of SEQ ID NOs: 1 to 41, a biologically active fragment of any one thereof, or an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs: I to 41, wherein the polypeptide is an olfactory receptor.
The present inventors have identified olfactory receptors that are highly expressed in female insects. Accordingly, in a further aspect the present invention provides a substantially purified and/or recombinant polypeptide comprising amino acids having a sequence as provided in any one of SEQ ID NOs: 12, 23, 38, 39, and 41, a biologically active fragment of any one thereof, or an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs: 12, 23, 38, 39, 40 and 41, wherein the polypeptide is a female specific olfactory receptor.
Preferably, the polypeptide comprises an amino acid sequence which is at least 90% identical to any one or more of SEQ ID NOs: 12, 23, 38, 39, 40 and 41.
Preferably, the polypeptide can be purified from a Lepidopteran.
In an embodiment, the polypeptide is fused to at least one other polypeptide.
The at least one other polypeptide may be, for example, a polypeptide that enhances the stability of a polypeptide of the present invention, a polypeptide that assists in the purification of the fusion protein, or a label that assists in the detection of intracellular signalling by the receptor upon ligand binding.
In a further embodiment, the present invention provides an isolated and/or exogenous polynucleotide comprising nucleotides having a sequence as provided in or complementary to any one of SEQ ID NOs: 42 to 82, a sequence which is at least 50% identical to any one or more of SEQ ID NOs: 42 to 82, a sequence which hybridizes to any one or more of SEQ ID NOs: 42 to 82, or a sequence which encodes a polypeptide of the invention.

Preferably, the polynucleotide comprises nucleotides having a sequence which hybridizes to one or more of SEQ ID NOs: 42 to 82 under stringent conditions.
More preferably, the polynucleotide comprises nucleotides having a sequence which hybridizes to one or more of SEQ ID NOs: 53, 64, 79, 80, 81 and 82 under stringent conditions.
Preferably, the polynucleotide encodes an olfactory receptor.
In an embodiment, the polynucleotide encodes a female specific olfactory receptor.
Also provided is an oligonucleotide which comprises at least 19 contiguous nucleotides of a polynucleotide of the invention.
In a further aspect, the present invention provides an isolated and/or exogenous polynucleotide which, when present in a cell of an insect, interferes with chemosensory perception of the insect when compared to a cell of an insect that lacks said polynucleotide, wherein the polynucleotide comprises nucleotides having a sequence as provided in or complementary to any one of SEQ ID NOs: 42 to 82, a sequence which is at least 50% identical to any one or more of SEQ ID NOs: 42 to 82, a sequence which hybridizes to any one or more of SEQ ID NOs: 42 to 82, a sequence which encodes a polypeptide of the invention, and/or which comprises an oligonucleotide of the invention.
Examples of such polynucleotide include, but are not limited to, an antisense polynucleotide, a catalytic polynucleotide and a double stranded RNA.
In an embodiment, the polynucleotide is a catalytic polynucleotide capable of cleaving a polynucleotide of the invention.
In another embodiment, the polynucleotide is a double stranded RNA
(dsRNA) molecule comprising an oligonucleotide of the invention, wherein the portion of the molecule that is double stranded is at least 19 basepairs in length and comprises said oligonucleotide.
In a further aspect, the present invention provides a vector comprising or encoding the polynucleotide of the invention.
Preferably, the polynucleotide, or sequence encoding the polynucleotide, is operably linked to a promoter.
In another aspect, the present invention provides a host cell comprising at least one polynucleotide of the invention, and/or at least one vector of the invention.
In yet another aspect, the present invention provides a process for preparing a polypeptide of the invention, the process comprising cultivating a host cell of the invention encoding said polypeptide, or a vector of the invention encoding said polypeptide, under conditions which allow expression of the polynucleotide encoding the polypeptide, and recovering the expressed polypeptide.

Also provided is an antibody which specifically binds a polypeptide of the invention.
In yet another aspect, the present invention provides a composition comprising a polypeptide of the invention, a polynucleotide of the invention, a vector of the 5 invention, a host of the invention and/or an antibody of the invention, and one or more acceptable carriers.
In another aspect, the present invention provides a kit comprising a polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, a host of the invention, an antibody of the invention, and/or a composition of the invention.
In another aspect, the present invention provides a method of identifying a molecule that binds to a polypeptide of the invention, the method comprising:
i) contacting a polypeptide of the invention with a candidate compound, ii) determining whether the compound binds the polypeptide.
In a further aspect, the present invention provides a method of identifying a molecule that binds to a polypeptide of the invention, the method comprising:
a) exposing a polypeptide of the invention to a binding partner which binds the polypeptide, and a candidate agent, and b) assessing the ability of the candidate agent to compete with the binding partner for binding to the polypeptide.
In one embodiment, the polypeptide comprises amino acids having a sequence as provided in SEQ ID NO: 12, an amino acid sequence which is at least 50%
identical to SEQ ID NO:12 or a biologically active fragment thereof, wherein the binding partner is linalool.
In another embodiment, the polypeptide comprises amino acids having a sequence as provided in SEQ ID NO:38, an amino acid sequence which is at least 50% identical to SEQ ID NO:38 or a biologically active fragment thereof, wherein the binding partner is 2-phenylethanol, benzoic acid, benzaldehyde, ethyl benzoate and/or methyl benzoate.
In one embodiment, the polypeptide comprises amino acids having a sequence as provided in SEQ ID NO:40, an amino acid sequence which is at least 50%
identical to SEQ ID NO:40 or a biologically active fragment thereof, wherein the binding partner is benzoic acid.
In an embodiment, the binding partner is an antibody or antigen-binding fragment thereof.
In a further embodiment, the binding partner is detectably labelled.
In yet another aspect, the present invention provides a method of identifying a molecule that binds to a polypeptide of the invention, the method comprising:

= 6 i) contacting a protein complex comprising a polypeptide of the invention with a candidate compound, ii) determining whether the compound binds the complex.
Preferably, the polypeptide is expressed in a cell.
Preferably, the polypeptide spans the cell membrane.
In an embodiment, the cell is an insect cell. Preferably, the insect cell is an olfactory receptor neuron. Preferably, the insect cell is a Lepidopteran cell.
The cell may be in vitro or in vivo.
In a further aspect, the present invention provides a method of identifying a molecule that modulates the activity of a polypeptide of the invention, the method comprising:
i) contacting a cell comprising a polypeptide of the invention with a candidate compound, ii) determining whether the compound modulates a physiologic activity of the cell.
In a further aspect, the present invention provides a method of identifying a molecule that modulates the activity of a polypeptide of the invention, the method comprising:
i) contacting a first cell comprising a polypeptide of the invention with a candidate compound, ii) contacting a second cell lacking the polypeptide with the candidate compound, and iii) determining whether the compound modulates a physiologic activity in the first or second cell, wherein the first and second cell are the same cell type, and wherein a compound that modulates a physiologic activity in the first cell but not the second cell is a modulator of the polypeptide.
Preferably, the cell is a cell of an organism.
Preferably, the first cell and second cell are cells of the same cell type from two different individuals of an organism of the same species.
Preferably, the organism is a Lepidopteran.
In an embodiment, the physiologic activity is determined by analysing a behavioural activity of the organism.
In another embodiment, the physiologic activity is G-protein activity.
Preferably, G-protein activity is determined by measuring calcium ion and/or cyclic AMP concentration in the cell.
In a further embodiment, the physiologic activity is determined using an electroolfactogram.

In yet another aspect, the present invention provides a method of screening for a compound that modulates the activity of a polypeptide of the invention, the method comprising using the structural coordinates of a crystal of the polypeptide to computationally evaluate a candidate compound for its ability to bind to the polypeptide.
Preferably, the compound is an odorant.
In one embodiment, the compound is an antagonist of the physiologic activity.
In another embodiment, the compound is an agonist of the physiologic activity.
Also provided is a compound identified using a method of the invention.
In a further aspect, the present invention provides a method for controlling an insect pest, the method comprising exposing the insect pest to an antagonist of the invention.
In another aspect, the present invention provides a method for controlling an insect pest, the method comprising exposing the insect pest to an agonist of the invention.
In yet a further aspect, the present invention provides a biosensor comprising a polypeptide of the invention.
In another aspect, the present invention provides a transgenic non-human animal comprising an exogenous polynucleotide, the polynucleotide encoding at least one polypeptide of the invention.
In a further aspect, the present invention provides a transgenic non-human animal comprising an exogenous polynucleotide of the invention, and/or a polynucleotide encoding therefor.
In yet another aspect, the present invention provides a transgenic plant comprising an exogenous polynucleotide, the polynucleotide encoding at least one polypeptide of the invention.
In a further aspect, the present invention provides a transgenic plant comprising an exogenous polynucleotide of the invention, and/or a polynucleotide encoding therefor.
In yet another aspect, the present invention provides a method for controlling an insect pest, the method comprising delivering to the insect a polynucleotide of the invention, and/or a polynucleotide encoding therefor.
In an embodiment, the polynucleotide is delivered by exposing the insect to a transgenic plant of the invention, wherein the insect eats the plant.
In a further aspect, the present invention provides a method of controlling female insect pests, the method comprising exposing the female insect pests to a ligand which binds a polypeptide comprising amino acids having a sequence as provided in any one of SEQ ID NOs: 12, 23, 38, 39, 40 and 41, a biologically active fragment of any one thereof, or an amino acid sequence which is at least 50%
identical to any one or more of SEQ ID NOs: 12, 23, 38, 39, 40 and 41.
In an embodiment, the ligand disrupts mating.
Examples of such ligands include, but are not limited to, linalool, 2-phenylethanol, benzoic acid, benzaldehyde, ethyl benzoate and methyl benzoate.
In a further embodiment, the insect pest is a Lepidopteran.
As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1 A) Gene structure of five female-biased silkworm Ors, BmOr19, 30 and 47. Each possesses the typical short last exon with a phase 0 intron/exon boundary.
Solid blocks represent exons, dashed lines denote intron sequence, a break in the line indicates that the sequence was not contiguous. The phase of each intron/exon boundary is indicated numerically above each intron. B) Protein alignment of five female-biased silkworm Ors with related Ors from Drosophila melanogaster (DmOr46a, AAF58834.3) and Anopheles gambiae (AgOrl, AAL35506). The greatest amino acid similarity occurs at the C-terminus of the protein sequence, particularly the typical Ser (sometimes Thr) residue in the seventh codon position of the last exon.
Figure 2. Neighbor-joining (corrected distance) phylogenetic tree of the Bombyx mori and Heliothis virescens Ors identified to date, rooted with lepidopteran members of the D. melanogaster Or83b lineage, BmOr2, HvOr2 and Helicoverpa zea Or2 (AAX14773). Krieger et al., (2004) reported 18 Or and 3 Gr H. virescens genes that were collectively referred to as chemoreceptors (Crs). Accession numbers for the silkworm Ors are listed in Table 2. Percentage bootstrap support (1000 replicates) is indicated at significant branch points. With the exception of BmOr9 and HvOr6, all members of the pheromone receptor subfamily are expresses at higher ratios in male compared to female adult moth antennae (Table 3, Figure 3, Sakurai et al., 2004;
Krieger et al., 2005; Nakagawa et al., 2005).

., ~ 9 Figure 3. Silkworm Or gene expression levels in female and male moth antennae determined by quantitative real-time PCR. Expression levels relative to the control gene BmRPS3 were calculated using the equation 2- CT (Livak and Schmittgen, 2001) and reported on a loglo scale. Abdomen tissue that lacks olfactory sensilla was included as a control reference and values were rounded up to a minimum of 10-relative to BmRPS3 for presentation. Ors whose expression was not detected in adult moth antennae (BmOr2O, 21, 22, 25 and 42) are not included in the figure.

Figure 4. Silkworm Or gene expression levels in the antennae of individual moths determined by quantitative real-time PCR, n = four females and four males.
Expression levels relative to the control gene BmRPS3 were calculated using the equation 2" CT (Livak and Schmittgen, 2001) and reported on a loglo scale, values were rounded up to a minimum of 10-4 relative to BmRPS3 for presentation.
Statistical significance of differences in expression levels (CT value) between female and male antennae was analyzed for each Or gene by nested Analysis of Variance (three technical replicates nested within each biological replicate).
Asterisks indicate statistically significant differences, *** indicates p < 0.001 and **
indicates p < 0.01.
Bars on each column represent the lower and upper 95% confidence interval for the mean expression value.
Figure 5. Activation of BmOrl9 signaling by linalool.

Figure 6. Activation of BmOr45 signaling by 2-phenylethanol.
Figure 7. Activation of BmOr45 signaling by benzaldehyde.
Figure 8. Activation of BmOr45 signaling by benzoic acid.
Figure 9. Activation of BmOr47 signaling by benzoic acid.
KEY TO THE SEQUENCE LISTING
SEQ ID NO:1 - Polypeptide sequence of BmOr8.
SEQ ID NO:2 - Partial polypeptide sequence of BmOr9.
SEQ ID NO:3 - Polypeptide sequence of BmOr10.
SEQ ID NO:4 - Polypeptide sequence of BmOrl 1.
SEQ ID NO:5 - Polypeptide sequence of BmOrl2.
SEQ ID NO:6 - Partial polypeptide sequence of BmOrl3.
SEQ ID NO:7 - Polypeptide sequence of BmOr14.

SEQ ID NO:8 - Polypeptide sequence of BmOr15.
SEQ ID NO:9 - Polypeptide sequence of BmOr16.
SEQ ID NO: 10 - Polypeptide sequence of BmOrl7.
SEQ ID NO:11 - Partial polypeptide sequence of BmOr18.
5 SEQ ID NO:12 - Polypeptide sequence of BmOr19.
SEQ ID NO: 13 - Polypeptide sequence of BmOr20.
SEQ ID NO:14 - Polypeptide sequence of BmOr21.
SEQ ID NO: 15 - Polypeptide sequence of BmOr22.
SEQ ID NO: 16 - Polypeptide sequence of BmOr23.
10 SEQ ID NO: 17 - Polypeptide sequence of BmOr24.
SEQ ID NO: 18 - Polypeptide sequence of BmOr25.
SEQ ID NO: 19 - Polypeptide sequence of BmOr26.
SEQ ID NO:20 - Polypeptide sequence of BmOr27.
SEQ ID NO:21 - Polypeptide sequence of BmOr28.
SEQ ID NO:22 - Polypeptide sequence of BmOr29.
SEQ ID NO:23 - Polypeptide sequence of BmOr30.
SEQ ID NO:24 - Polypeptide sequence of BmOr3 1.
SEQ ID NO:25 - Partial polypeptide sequence of BmOr32.
SEQ ID NO:26 - Polypeptide sequence of BmOr33.
SEQ ID NO:27 - Polypeptide sequence of BmOr34.
SEQ ID NO:28 - Polypeptide sequence of BmOr35.
SEQ ID NO:29 - Polypeptide sequence of BmOr36.
SEQ ID NO:30 - Polypeptide sequence of BmOr37.
SEQ ID NO:31 - Polypeptide sequence of BmOr38.
SEQ ID NO:32 - Polypeptide sequence of BmOr39.
SEQ ID NO:33 - Partial polypeptide sequence of BmOr4O.
SEQ ID NO:34 - Polypeptide sequence of BmOr4l.
SEQ ID NO:35 - Polypeptide sequence of BmOr42.
SEQ ID NO:36 - Partial polypeptide sequence of BmOr43.
SEQ ID NO:37 - Partial polypeptide sequence of BmOr44.
SEQ ID NO:38 - Polypeptide sequence of BmOr45.
SEQ ID NO:39 - Partial polypeptide sequence of BmOr46.
SEQ ID NO:40 - Polypeptide sequence of BmOr47.
SEQ ID NO:41 - Partial polypeptide sequence of BmOr48.
SEQ ID NO:42 - Nucleotide sequence encoding BmOr8.
SEQ ID NO:43 - Nucleotide sequence encoding BmOr9.
SEQ ID NO:44 - Nucleotide sequence encoding BmOr10.
SEQ ID NO:45 - Nucleotide sequence encoding BmOrl 1.

SEQ ID NO:46 - Nucleotide sequence encoding BmOr12.
SEQ ID NO:47 -Nucleotide sequence encoding BmOr13.
SEQ ID NO:48 - Nucleotide sequence encoding BmOrl4.
SEQ ID NO:49 - Nucleotide sequence encoding BmOr15.
SEQ ID NO:50 - Nucleotide sequence encoding BmOr16.
SEQ ID NO:51 -Nucleotide sequence encoding BmOrl7.
SEQ ID NO:52 - Nucleotide sequence encoding BmOrl8.
SEQ ID NO:53 - Nucleotide sequence encoding BmOr19.
SEQ ID NO:54 - Nucleotide sequence encoding BmOr2O.
SEQ ID NO:55 - Nucleotide sequence encoding BmOr21.
SEQ ID NO:56 - Nucleotide sequence encoding BmOr22.
SEQ ID NO:57 - Nucleotide sequence encoding BmOr23.
SEQ ID NO:58 - Nucleotide sequence encoding BmOr24.
SEQ ID NO:59 - Nucleotide sequence encoding BmOr25.
SEQ ID NO:60 - Nucleotide sequence encoding BmOr26.
SEQ ID NO:61 - Nucleotide sequence encoding BmOr27.
SEQ ID NO:62 - Nucleotide sequence encoding BmOr28.
SEQ ID NO:63 - Nucleotide sequence encoding BmOr29.
SEQ ID NO:64 - Nucleotide sequence encoding BmOr3O.
SEQ ID NO:65 - Nucleotide sequence encoding BmOr3 1.
SEQ ID NO:66 - Nucleotide sequence encoding BmOr32.
SEQ ID NO:67 - Nucleotide sequence encoding BmOr33.
SEQ ID NO:68 - Nucleotide sequence encoding BmOr34.
SEQ ID NO:69 - Nucleotide sequence encoding BmOr35.
SEQ ID NO:70 - Nucleotide sequence encoding BmOr36.
SEQ ID NO:71 - Nucleotide sequence encoding BmOr37.
SEQ ID NO:72 - Nucleotide sequence encoding BmOr38.
SEQ ID NO:73 - Nucleotide sequence encoding BmOr39.
SEQ ID NO:74 - Nucleotide sequence encoding BmOr4O.
SEQ ID NO: 75 - Nucleotide sequence encoding BmOr4 1.
SEQ ID NO:76 - Nucleotide sequence encoding BmOr42.
SEQ ID NO:77 - Nucleotide sequence encoding BmOr43.
SEQ ID NO:78 - Nucleotide sequence encoding BmOr44.
SEQ ID NO:79 - Nucleotide sequence encoding BmOr45.
SEQ ID NO:80 - Nucleotide sequence encoding BmOr46.
SEQ ID NO:81 - Nucleotide sequence encoding BmOr47.
SEQ ID NO:82 - Nucleotide sequence encoding BmOr48.
SEQ ID NO's 83 to 178 - Oligonucleotide primers.

SEQ ID NO: 179 - Polypeptide sequence of DmOr46a (Genbank: AAF58834.3).
SEQ ID NO: 180 - Polypeptide sequence of AgOrl (Genbank: AAL35506).
DETAILED DESCRIPTION OF THE INVENTION
General Techniques and Definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, receptor biology, biosensors, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL
Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley &
Sons (including all updates until present).
As used herein, the term "olfactory receptor", "insect olfactory receptor", "odorant receptor" or "insect odorant receptor" or variations thereof when used in relation to the polypeptides of the invention refers to a polypeptide which, when present in a cell of an insect, is involved in chemosensory perception of the insect.
Preferably, the insect is a Lepidopteran. Preferably, the cell is a neuron, more preferably a neuron cell in the antenna of the insect. Furthermore, the term "olfactory receptor" or "insect olfactory receptor" when used in relation to the polypeptides of the invention refers to a polypeptide which binds an odorant ligand resulting in a physiologic response. Furthermore, the term "olfactory receptor", "insect olfactory receptor", "odorant receptor" or "insect odorant receptor" or variations thereof is used broadly to refer to a polypeptide of the invention in isolation, or as a complex with at least one other protein, wherein said other protein is typically also a receptor molecule involved in chemosensory perception of an insect. In one embodiment, the at least one other protein is a different olfactory receptor sub-unit when compared to the polypeptides of the invention.
As used herein, the term "female specific" means that the polypeptide is produced at a higher level in adult females than adult males. Preferably, the female:male expression in antennae is at least 3.0, more preferably 5Ø This term also includes mutants of naturally occurring female specific receptors which have been mutated. Such mutants are at least 95%, more preferably at least 97%, and more preferably at least 99% identical to the naturally produced polypeptide.
"Polynucleotide" refers to a oligonucleotide, nucleic acid molecule or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity defined herein.
"Operably linked" as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence.
For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell and/or in a cell-free expression system.
Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
Compositions of the present invention may include an "acceptable carrier".
Examples of such acceptable carriers include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. The exact nature of the "acceptable carrier" will depend on the use of the composition. Considering the uses described herein, and the nature of the component of the invention in the composition, the skilled person can readily determine suitable a "acceptable carrier(s)" for a particular use.

Polypeptides By "substantially purified polypeptide" or "purified" we mean a polypeptide that has generally been separated from the lipids, nucleic acids, other polypeptides, and other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated.
The term "recombinant" in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate compared to its native state. In one embodiment the cell is a cell that does not naturally produce the polypeptide. However, the cell may be a cell which comprises a non-endogenous gene that causes an altered, preferably increased, amount of the polypeptide to be produced. A recombinant polypeptide of the invention includes polypeptides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.
The terms "polypeptide" and "protein" are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors.
The terms "proteins" and "polypeptides" as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the invention as described herein.
The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids.
More preferably, the query sequence is at least 50 amino acids in length, and the GAP
analysis aligns the two sequences over a region of at least 50 amino acids.
More preferably, the query sequence is at least 100 amino acids in length and the GAP
analysis aligns the two sequences over a region of at least 100 amino acids.
Even more preferably, the query sequence is at least 250 amino acids in length and the GAP
analysis aligns the two sequences over a region of at least 250 amino acids.
Most preferably, the two sequences are aligned over their entire length.
As used herein a "biologically active" fragment is a portion of a polypeptide of the invention which maintains a defined activity of the full-length polypeptide, namely be able to act as an olfactory receptor. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, biologically active fragments are at least 100, more preferably at least 200, and even more preferably at least 350 amino acids in length.
With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments.

Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 5 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more 10 preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
Amino acid sequence mutants of the polypeptides of the present invention can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the 15 present invention, or by in vitro synthesis of the desired polypeptide.
Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
Mutant (altered) polypeptides can be prepared using any technique known in the art. For example, a polynucleotide of the invention can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. In another example, the polynucleotides of the invention, including genes encoding therefor, are subjected to DNA shuffling techniques as broadly described by Harayama (1998). These DNA shuffling techniques may include genes related to those of the present invention, such as homologous genes encoding olfactory receptors from many different Lepidopterans.
Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they have olfactory receptor activity.
In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical (see, for example, the protein alignments provided herein). These positions may be important for biological activity.
These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".

Table 1. Exemplary substitutions Original Exemplary Residue Substitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; his Asp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, ala His (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; phe L s (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S) thr Thr (T) ser Trp (W) tyr Tyr (Y) t ; phe Val (V) ile; leu; met; phe; ala In a preferred embodiment a mutant/variant polypeptide has one or two or three or four or five conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 1. Sites of particular interest to alter are those which are not conserved between two, three or more of the polypeptides described herein. Examples of such conserved amino acids are provided in Figure 1B. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell or cell free system.
Furthermore, if desired, unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the polypeptides of the present invention. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, (3-alanine, fluoro-amino acids, designer amino acids such as (3-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogues in general.
Also included within the scope of the invention are polypeptides of the present invention which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide of the invention.
Polypeptides of the present invention can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides. In one embodiment, an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention.
Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates.
Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
Polynucleotides and Oligonucleotides By an "isolated polynucleotide", including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, dsRNA or otherwise, we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state. Preferably, the isolated polynucleotide is at least 60%
free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. Furthermore, the term "polynucleotide" is used interchangeably herein with the term "nucleic acid".
The term "exogenous" in the context of a polynucleotide refers to the polynucleotide when present in a cell, or in a cell-free expression system, in an altered amount compared to its native state. In one embodiment, the cell is a cell that does not naturally comprise the polynucleotide. However, the cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered, preferably increased, amount of production of the encoded polypeptide. An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components. The exogenous polynucleotide (nucleic acid) can be a contiguous stretch of nucleotides existing in nature, or comprise two or more contiguous stretches of nucleotides from different sources (naturally occurring and/or synthetic) joined to form a single polynucleotide. Typically such chimeric polynucleotides comprise at least an open reading frame encoding a polypeptide of the invention operably linked to a promoter suitable of driving transcription of the open reading frame in a cell of interest.
The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, the query sequence is at least nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Most preferably, the two sequences are aligned over their entire length.
With regard to the defined polynucleotides, it will be appreciated that %
identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that a polynucleotide of the invention comprises a sequence which is at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
Polynucleotides of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the nucleic acid).
The term "stringent hybridization conditions" or "stringent conditions" and the like as used herein refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an polynucleotide or oligonucleotide. Nucleic acid hybridization parameters may be found in references which compile such methods, Sambrook, et al., (supra), and Ausubel, et al., (supra).
For example, stringent hybridization conditions, as used herein, can refer to hybridization at 65 C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02%
polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH2PO4 (pH7), 0.5%
SDS, 2 mM EDTA) and washing twice in 0.2xSSC, 0.1% SDS at 65 C, with each wash step being about 30 min. Alternatively, the nucleic acid and/or oligonucleotides (which may also be referred to as "primers" or "probes") hybridize to the region of the an insect genome of interest, such as the genome of a Lepidopteran, under conditions used in nucleic acid amplification techniques such as PCR.
Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. Although the terms polynucleotide and oligonucleotide have overlapping meaning, oligonucleotide are typically relatively short single stranded molecules. The = 20 minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a target nucleic acid molecule. Preferably, the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 25 nucleotides in length.
Usually, monomers of a polynucleotide or oligonucleotide are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a relatively short monomeric units, e.g., 12-18, to several hundreds of monomeric units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate.
The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, or primers to produce nucleic acid molecules. Oligonucleotide of the present invention used as a probe are typically conjugated with a detectable label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.
Probes and/or primers can be used to clone further homologues of the olfactory receptor cDNAs described herein from other Lepidopteran species. Further hybridization techniques known in the art can also be used to screen genomic or cDNA libraries for such homologues. In addition, techniques such as 5' and 3' RACE
can be used to determine the entire open reading frame, and the corresponding full length protein sequence, of any partial sequences provided herein. In light of the present disclosure, the skilled person would find such techniques routine.
Antisense Polynucleotides The term "antisense polynucleotide" shall be taken to mean a DNA or RNA, or combination thereof, molecule that is complementary to at least a portion of a specific mRNA molecule encoding a polypeptide of the invention and capable of interfering with a post-transcriptional event such as mRNA translation. The use of antisense methods is well known in the art (see for example, G. Hartmann and S. Endres, Manual of Antisense Methodology, Kluwer (1999)).
An antisense polynucleotide of the invention hybridises under physiological conditions to a target polynucleotide (which is fully or partially single stranded), and thus are at least capable of forming a double stranded polynucleotide with mRNA
encoding a protein, such as those proteins provided in SEQ ID NOs: I to 41, under normal conditions in a cell.

Antisense molecules may include sequences that correspond to the structural genes or for sequences that effect control over the gene expression or splicing event.
For example, the antisense sequence may correspond to the targeted coding region of the genes of the invention, or the 5'-untranslated region (UTR) or the 3'-UTR
or combination of these. It may be complementary in part to intron sequences, which may be spliced out during or after transcription, preferably only to exon sequences of the target gene. In view of the generally greater divergence of the UTRs, targeting these regions provides greater specificity of gene inhibition.
The length of the antisense sequence should be at least 19 contiguous nucleotides, preferably at least 50 nucleotides, and more preferably at least 100, 200, or 500 nucleotides. The full-length sequence complementary to the entire gene transcript may be used. The length is most preferably 100-2000 nucleotides.
The degree of identity of the antisense sequence to the targeted transcript should be at least 90% and more preferably 95-100%. The antisense RNA molecule may of course comprise unrelated sequences which may function to stabilize the molecule.

Catal tiy c Polynucleotides The term catalytic polynucleotide/nucleic acid refers to a DNA molecule or DNA-containing molecule (also known in the art as a "deoxyribozyme") or an RNA
or RNA-containing molecule (also known as a "ribozyme") which specifically recognizes a distinct substrate and catalyzes the chemical modification of this substrate. The nucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T
(and U for RNA).
Typically, the catalytic nucleic acid contains an antisense sequence for specific recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic activity (also referred to herein as the "catalytic domain"). The types of ribozymes that are particularly useful in this invention are the hammerhead ribozyme (Haseloff and Gerlach, 1988, Perriman et al., 1992) and the hairpin ribozyme (Shippy et al., 1999).
The ribozymes of this invention and DNA encoding the ribozymes can be chemically synthesized using methods well known in the art. The ribozymes can also be prepared from a DNA molecule (that upon transcription, yields an RNA
molecule) operably linked to an RNA polymerase promoter, e.g., the promoter for T7 RNA
polymerase or SP6 RNA polymerase. Accordingly, also provided by this invention is a nucleic acid molecule, i.e., DNA or cDNA, coding for a catalytic polynucleotide of the invention. When the vector also contains an RNA polymerase promoter operably linked to the DNA molecule, the ribozyme can be produced in vitro upon incubation with RNA polymerase and nucleotides. In a separate embodiment, the DNA can be inserted into an expression cassette or transcription cassette. After synthesis, the RNA molecule can be modified by ligation to a DNA molecule having the ability to stabilize the ribozyme and make it resistant to RNase.
As with antisense polynucleotides described herein, catalytic polynucleotides of the invention should also be capable of hybridizing a target nucleic acid molecule (for example an mRNA encoding a polypeptide provided as any one of SEQ ID NOs:
1 to 41) under "physiological conditions", namely those conditions within a cell (especially conditions in an insect cell such as a cell of a lepidpoteran).

RNA interference RNA interference (RNAi) is particularly useful for specifically inhibiting the production of a particular protein. Although not wishing to be limited by theory, Waterhouse et al., (1998) have provided a model for the mechanism by which dsRNA
can be used to reduce protein production. This technology relies on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest or part thereof, in this case an mRNA encoding a polypeptide according to the invention. Conveniently, the dsRNA can be produced from a single promoter in a recombinant vector or host cell, where the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. The design and production of suitable dsRNA
molecules for the present invention is well within the capacity of a person skilled in the art, particularly considering Waterhouse et al., (1998), Smith et al., (2000), WO
99/32619, WO 99/53050, WO 99/49029, and WO 01/34815.
In one example, a DNA is introduced that directs the synthesis of an at least partly double stranded RNA product(s) with homology to the target gene to be inactivated. The DNA therefore comprises both sense and antisense sequences that, when transcribed into RNA, can hybridize to form the double-stranded RNA
region.
In a preferred embodiment, the sense and antisense sequences are separated by a spacer region that comprises an intron which, when transcribed into RNA, is spliced out. This arrangement has been shown to result in a higher efficiency of gene silencing. The double-stranded region may comprise one or two RNA molecules, transcribed from either one DNA region or two. The presence of the double stranded molecule is thought to trigger a response from an endogenous plant system that destroys both the double stranded RNA and also the homologous RNA transcript from the target plant gene, efficiently reducing or eliminating the activity of the target gene.
The length of the sense and antisense sequences that hybridise should each be at least 19 contiguous nucleotides, preferably at least 30 or 50 nucleotides, and more preferably at least 100, 200, or 500 or 1000 nucleotides. The full-length sequence corresponding to the entire gene transcript may be used. The lengths are most preferably 100-2000 nucleotides. The degree of identity of the sense and antisense sequences to the targeted transcript should be at least 85%, preferably at least 90%
and more preferably 95-100%. The RNA molecule may of course comprise unrelated sequences which may function to stabilize the molecule. The RNA molecule may be expressed under the control of a RNA polymerase II or RNA polymerase III
promoter. Examples of the latter include tRNA or snRNA promoters.
Preferred small interfering RNA (`siRNA") molecules comprise a nucleotide sequence that is identical to about 19-23 contiguous nucleotides of the target mRNA.
Preferably, the target mRNA sequence commences with the dinucleotide AA, comprises a GC-content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the insect (preferably lepidopteran) in which it is to be introduced, e.g., as determined by standard BLAST search.

Recombinant Vectors One embodiment of the present invention includes a recombinant vector, which comprises at least one isolated polynucleotide molecule of the present invention, inserted into any vector capable of delivering the polynucleotide molecule into a host cell. Such vectors contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a transposon (such as described in US 5,792,294), a virus or a plasmid.
One type of recombinant vector comprises a polynucleotide molecule of the present invention operatively linked to an expression vector. The phrase operatively linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector includes a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, animal, and plant cells. Vectors of the invention can also be used to produce the polypeptide in a cell-free expression system, such systems are well known in the art.
In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription.
Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences.
Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A
variety of such transcription control sequences are known to those skilled in the art.
Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, nematode, plant or mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SPO1, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells.
Host Cells Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention, or progeny cells thereof. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.

Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention. Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides of the present invention or can be capable of producing such polypeptides after being transformed 5 with at least one polynucleotide molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite, nematode, arthropod, animal and plant cells. Examples of host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, 10 Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (e.g., COS-7) cells, and Vero cells. Further examples of host cells are E.
coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; BHK
cells;
MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic 15 mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK cells and/or HeLa cells.
Particularly preferred host cells are insect cells, especially lepidopteran cells, as well 20 as nematode cells such as C. elegans cells.
Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant 25 transcripts are translated, and the efficiency of post-translational modifications.
Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
Transgenic Plants The term "plant" refers to whole plants, plant organs (e.g. leaves, stems roots, etc), seeds, plant cells and the like. Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. Target plants include, but are not limited to, the following: cereals (wheat, barley, rye, oats, rice, sorghum and related crops); beet (sugar beet and fodder beet); pomes, stone fruit and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and black-berries); leguminous plants (beans, lentils, peas, soybeans); oil plants (rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts); cucumber plants (marrows, cucumbers, melons); fibre plants (cotton, flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit, mandarins);
vegetables (spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, paprika);
lauraceae (avocados, cinnamon, camphor); or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, turf, bananas and natural rubber plants, as well as ornamentals (flowers, shrubs, broad-leaved trees and evergreens, such as conifers).
Transgenic plants, as defined in the context of the present invention include plants (as well as parts and cells of said plants) and their progeny which have been genetically modified using recombinant techniques to cause production of at least one polypeptide and/or polynucleotide of the present invention in the desired plant or plant organ. Transgenic plants can be produced using techniques known in the art, such as those generally described in A. Slater et al., Plant Biotechnology -The Genetic Manipulation of Plants, Oxford University Press (2003), and P.
Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).
A polynucleotide of the present invention may be expressed constitutively in the transgenic plants during all stages of development. Depending on the use of the plant or plant organs, the polynucleotides may be expressed in a stage-specific manner. Furthermore, the polynucleotides may be expressed tissue-specifically.
Regulatory sequences which are known or are found to cause expression of a polynucleotide of interest in plants may be used in the present invention. The choice of the regulatory sequences used depends on the target plant and/or target organ of interest. Such regulatory sequences may be obtained from plants or plant viruses, or may be chemically synthesized. Such regulatory sequences are well known to those skilled in the art.
Constitutive plant promoters are well known. Further to previously mentioned promoters, some other suitable promoters include but are not limited to the nopaline synthase promoter, the octopine synthase promoter, CaMV 35S promoter, the ribulose-1,5-bisphosphate carboxylase promoter, Adhl-based pEmu, Actl, the SAM
synthase promoter and Ubi promoters and the promoter of the chlorophyll a/b binding protein. Alternatively it may be desired to have the transgene(s) expressed in a regulated fashion. Regulated expression of the polynucleotides is possible by placing the coding sequence under the control of promoters that are tissue-specific, developmental-specific, or inducible. Several tissue-specific regulated genes and/or promoters have been reported in plants. These include genes encoding the seed storage proteins (such as napin, cruciferin, (3-conglycinin, glycinin and phaseolin), zein or oil body proteins (such as oleosin), or genes involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase, and fatty acid desaturases (fad 2- 1)), and other genes expressed during embryo development (such as Bce4).
Particularly useful for seed-specific expression is the pea vicilin promoter.
Other useful promoters for expression in mature leaves are those that are switched on at the onset of senescence, such as the SAG promoter from Arabidopsis. A class of fruit-specific promoters expressed at or during anthesis through fruit development, at least until the beginning of ripening, is discussed in US 4,943,674. Other examples of tissue-specific promoters include those that direct expression in leaf cells following damage to the leaf (for example, from chewing insects), in tubers (for example, patatin gene promoter), and in fiber cells (an example of a developmentally-regulated fiber cell protein is E6 fiber.
Several techniques are available for the introduction of an expression construct containing a nucleic acid sequence of interest into the target plants. Such techniques include but are not limited to transformation of protoplasts using the calcium/polyethylene glycol method, electroporation and microinjection or (coated) particle bombardment. In addition to these so-called direct DNA transformation methods, transformation systems involving vectors are widely available, such as viral and bacterial vectors (e.g. from the genus Agrobacterium). After selection and/or screening, the protoplasts, cells or plant parts that have been transformed can be regenerated into whole plants, using methods known in the art. The choice of the transformation and/or regeneration techniques is not critical for this invention.
To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way to quantitate expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype.
The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.

Transgenic Non-Human Animals Techniques for producing transgenic animals are well known in the art. A
useful general textbook on this subject is Houdebine, Transgenic animals -Generation and Use (Harwood Academic, 1997).
Heterologous DNA can be introduced, for example, into fertilized mammalian ova. For instance, totipotent or pluripotent stem cells can be transformed by microinjection, calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means, the transformed cells are then introduced into the embryo, and the embryo then develops into a transgenic animal. In a highly preferred method, developing embryos are infected with a retrovirus containing the desired DNA, and transgenic animals produced from the infected embryo. In a most preferred method, however, the appropriate DNAs are coinjected into the pronucleus or cytoplasm of embryos, preferably at the single cell stage, and the embryos allowed to develop into mature transgenic animals.
Another method used to produce a transgenic animal involves microinjecting a nucleic acid into pro-nuclear stage eggs by standard methods. Injected eggs are then cultured before transfer into the oviducts of pseudopregnant recipients.
Transgenic animals may also be produced by nuclear transfer technology.
Using this method, fibroblasts from donor animals are stably transfected with a plasmid incorporating the coding sequences for a binding domain or binding partner of interest under the control of regulatory sequences. Stable transfectants are then fused to enucleated oocytes, cultured and transferred into female recipients.
Antibodies The invention also provides monoclonal or polyclonal antibodies to polypeptides of the invention or fragments thereof. Thus, the present invention further provides a process for the production of monoclonal or polyclonal antibodies to polypeptides of the invention.
The term "specifically binds" refers to the ability of the antibody to bind to at least one polypeptide of the present invention but not other known olfactory receptors.
In an embodiment, an antibody of the invention is an antagonist of an olfactory receptor of the invention.
As used herein, the term "epitope" refers to a region of a polypeptide of the invention which is bound by the antibody. An epitope can be administered to an animal to generate antibodies against the epitope, however, antibodies of the present invention preferably specifically bind the epitope region in the context of the entire polypeptide.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide of the invention.
Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals.
Monoclonal antibodies directed against polypeptides of the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced can be screened for various properties; i.e., for isotype and epitope affinity.
An alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs). This technique is well known in the art.
For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
In an embodiment, antibodies of the present invention are detectably labeled.
Exemplary detectable labels that allow for direct measurement of antibody binding include radiolabels, fluorophores, dyes, magnetic beads, chemiluminescers, colloidal particles, and the like. Examples of labels which permit indirect measurement of binding include enzymes where the substrate may provide for a coloured or fluorescent product. Additional exemplary detectable labels include covalently bound enzymes capable of providing a detectable product signal after addition of suitable substrate. Examples of suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art. Further exemplary detectable labels include biotin, which binds with high affinity to avidin or streptavidin;
fluorochromes (e.g., phycobiliproteins, phycoerythrin and allophycocyanins; fluorescein and Texas red), which can be used with a fluorescence activated cell sorter; haptens;
and the like.
5 Preferably, the detectable label allows for direct measurement in a plate luminometer, e.g., biotin. Such labeled antibodies can be used in techniques known in the art to detect polypeptides of the invention.

Identification of Compounds that Bind and/or Modulate the Activity Olfactory 10 Receptors The present invention provides screening methodologies useful in the identification of compounds which bind to and/or modulate the activity of the olfactory receptor genes, mRNA and proteins described herein. Such compounds will include molecules that agonize or antagonize olfactory receptor function.
15 Screening methodologies to identify compounds that bind and/or modulate the activity of olfactory receptors are known in the art. Such compounds include endogenous cellular components which interact with the identified genes and proteins in vivo. Thus, cell lysates or tissue homogenates may be screened for proteins or other compounds which bind to one of the olfactory receptor genes, mRNA or 20 proteins of the invention.
Alternatively, any of a variety of exogenous compounds, both naturally occurring and/or synthetic (e. g., libraries of small molecules or peptides), may be screened for binding capacity. Binding compounds can include, but are not limited to, other cellular proteins. Binding compounds can also include, but are not limited to, 25 peptides such as, for example, soluble peptides, including, but not limited to, Ig-tailed fusion peptides, antibodies such as those described herein, and small organic or inorganic molecules. Such compounds can include organic molecules (e. g., peptidomimetics) that bind to the receptor and either mimic the activity triggered by the natural odorant ligand (namely, agonists); as well as peptides, antibodies and other 30 organic compounds that mimic the receptor (or a portion thereof) and bind to and "neutralize" natural odorant ligand. Such compounds identified in a screen for binding to the receptor can be assayed for their effects on receptor signalling.
Particularly useful molecules that bind to and/or modulate olfactory receptor activity are small molecules, most preferably volatile small molecules, that function as odorants. The term "odorant" as employed herein refers to a molecule that has the potential to bind to an olfactory receptor. Equivalent terms employed herein include "odorant ligand", "odorant molecule" and "odorant compound". The term "binding"
or "interaction" as used herein with respect to odorant ligands refers to the interaction of ligands with the receptor polypeptide where the ligands may serve as either agonists and/or antagonists of a given receptor or receptor function. This effect may not be direct, but merely by altering the binding of an odorant receptor to another ligand. An odorant ligand may thus directly cause a perception of odor (an agonist), or may block the perception of odor (an antagonist). An odorant ligand may include, but is not limited to, molecules which interact with polypeptides involved in olfactory sensation.
Binding of a modulator (ligand) to a receptor of the invention can be examined in vitro with soluble or solid state reactions, using a full-length receptor molecule or a chimeric molecule such as an extracellular domain or transmembrane region, or combination thereof, of an receptor of the invention covalently linked to a heterologous signal transduction domain, or a heterologous extracellular domain and/or transmembrane region covalently linked to the transmembrane and/or cytoplasmic domain of an olfactory receptor. Furthermore, ligand-binding domains of the protein of interest can be used in vitro in soluble or solid state reactions to assay for ligand binding. In numerous embodiments, a chimeric receptor will be made that comprises all or part of an olfactory receptor polypeptide, as well an additional sequence that facilitates the localization of the olfactory receptor to the membrane, such as a rhodopsin, e. g., an N-terminal fragment of a rhodopsin protein.
Ligand binding to an olfactory receptor of the invention, a domain, or chimeric protein (also referred to herein as a fusion protein) can be tested in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles.
Binding of a modulator can be tested using, e. g., changes in spectroscopic characteristics (e. g., fluorescence, absorbance, refractive index) hydrodynamic (e. g., shape), chromatographic, or solubility properties. These assays may involve displacing a radioactively or fluorescently labeled ligand, and measuring changes in intrinsic fluorescence or changes in proteolytic susceptibility, etc.
Methods for screening odorant compounds using olfactory receptors in neuronal cells are known in the art (WO 98/50081; Duchamp-Viret et al., 1999;
Sato et al., 1994; Malnic et al., 1999; Zhao et al., 1998). There are also methods which can be employed to screen odorant compounds which do not require neuronal cells and that are known in the art (US 5,798, 275; Kiefer et al., 1996; Krautwurst et al., 1998).
The invention provides methods and compositions for expressing the olfactory receptors of the invention in cells to screen for odorants that can specifically bind an olfactory receptor of the invention, and for determining the effect (e. g., biochemical or electrophysiological) of such binding on cell physiology.
Any cell expression system can be used, e. g., insect or mammalian (for example HEK293, CHO or COS cells) cell expression systems. Cells that normally express olfactory receptors can be used, particularly to study the physiological effect of an odorant on a cell. Isolation and/or culturing of such cells and their transformation with the olfactory receptor-expressing sequences of the invention can be done with routine methods (Vargas, 1999; Coon et al., 1989).
Several methods of measuring G-protein activity are known to those of skill in the art and can be used in conjunction with the methods of the present invention, including but not limited to measuring calcium ion or cyclic AMP concentration in the cells. Such methods are described in Howard et al., (2001), Krautwurst et al., (1999), Chandrashekar et al., (2000), Oda et al., (2000) and Kiely et al., (2007).
To evaluate electrophysiologic effects of ligand binding to cell-expressed olfactory receptor of the invention, patch-clamping of individual cells can be done.
Patch-clamp recordings of the olfactory receptor cell membrane can measure membrane conductances. Some conductances are gated by odorants in the cilia and depolarize the cell through cAMP-or IP3-sensitive channels, depending on the species. Other conductances are activated by membrane depolarization and/or an increased intracellular Ca2+ concentration (Trotier, 1994).
Changes in calcium ion levels in the cell after exposure of the cell to known or potential odorant/ligands can be detected by a variety of means. For example, cells can be pre-loaded with reagents sensitive to calcium ion transients.
Techniques for the measurement of calcium transients are known in the art. For example, Kashiwayanagi (1996) measured both of inositol 1,4,5-trisphosphate induces inward currents and Ca2+ uptake in frog olfactory receptor cells.
In certain specific embodiments, intracellular calcium concentration is measured in the screening assays of the instant application by using a Fluorometric Imaging Plate Reader ("FLIPR") system (Molecular Devices, Inc.), which provides the advantages of automated, high-throughput screening, see also Sullivan et al., "Measurement of [Ca2+] i using the fluorometric imaging plate reader (FLIPR)", p.
125-136, Calcium Signaling Protocols D. G. Lambert, (editor), Humana Press (1999);
or in US 6,004, 808, which employs Fura-PE3 (Molecular Probes, Inc., Eugene, OR) as a stain of calcium ions.
Other physiologic activity mechanisms can also be measured, e. g., plasma membrane homeostasis parameters (including lipid second messengers), and cellular pH changes (see, e. g., Silver, 1998).
Alternatively, in vitro synthesised mRNA coding for a polypeptide of the invention can be injected into Xenopus oocytes allowing electrophysiological or calcium imaging of odorant-driven cell excitation.
A typical principle of the assays used to identify compounds that bind to olfactory receptors of the invention involves preparing a reaction mixture of said receptor and a test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay involves attaching the receptor or the test substance onto a solid phase and detecting receptor/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the receptor can be anchored onto a solid surface, and the test compound, which is not anchored, can be labeled, either directly or indirectly.
In practice, microtiter plates can conveniently be utilized as the solid phase.
The anchored component can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface. The surfaces can be prepared in advance and stored.
In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e. g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways.
Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e. g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e. g., using an immobilized antibody specific for an olfactory receptor of the invention or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
High throughput screening assays can also be used to identify compounds that bind and/or modulate an olfactory receptor of the invention. In the high throughput assays of the invention, it is possible to screen up to several thousand different ligands or modulators in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.
g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 1000 to about 1500 different compounds. It is possible to assay several plates per day. More recently, microfluidic approaches to reagent manipulation have been developed.
Additionally, methods can be employed which result in the simultaneous identification of genes which encode proteins interacting with an olfactory receptor of the invention. These methods include, for example, probing expression libraries with labeled polypeptide of the invention, using this protein in a manner similar to the well known technique of antibody probing of ?,gtl l libraries.
One method which detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration purposes only and not by way of limitation. One version of this system has been described (Chien et al., 1991) and is commercially available from Clontech (Palo Alto, CA). Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to a known protein, in this case, a polypeptide of the invention, and the other consists of the activator protein's activation domain fused to an unknown protein that is encoded by a cDNA, preferably an insect (more preferably a Lepidopteran) antennal or maxillary palp cDNA, which has been recombined into this plasmid as part of a cDNA library. The plasmids are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e. g., lacZ) whose regulatory region contains the transcription activator's binding sites.
Either hybrid protein alone cannot activate transcription of the reporter gene, the DNA-binding domain hybrid cannot because it does not provide activation function, and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter polypeptide.

Protein-Structure Based Desi n~gonists and Antagonists Computer modeling and searching technologies permit identification of compounds that can bind olfactory receptors of the invention, including compounds that can modulate olfactory receptor activity. The identification of such a compound may also allow the active sites or regions of the receptor to be identified.
Such active sites might typically be odorant ligand binding sites, such as the interaction domains of odorant ligands with the receptor.
The three dimensional geometric structure of olfactory receptor or the active site thereof can be determined. This can be done by known methods, including X-ray ' 35 crystallography, which can determine a complete molecular structure. Solid or liquid phase NMR can also be used to determine certain intra-molecular distances within the active site and/or in the odorant ligand/receptor complex. Any experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures may be measured with a complexed odorant ligand, natural or artificial, which may increase the accuracy of the receptor structure, or active site structure, that is determined.
Methods of computer based numerical modeling can be used to complete the structure (e. g., in embodiments wherein an incomplete or insufficiently accurate structure is determined) or to improve its accuracy. Any method recognized in the art may be used, including, but not limited to, parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
The three-dimensional structure of an olfactory receptor of the invention can be used to identify antagonists or agonists through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (Dunbrack et al., 1997). Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of a candidate compound to the polypeptide. Generally the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force) the more potent the potential agonist or antagonist will be since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential agonist or antagonist the more likely that it will not interfere with other proteins.
Initially a potential compound could be obtained, for example, using methods of the invention such as by screening a random peptide library produced by a recombinant bacteriophage or a chemical library. A compound selected in this manner could be then be systematically modified by computer modeling programs until one or more promising potential compounds are identified.
Such computer modeling allows the selection of a finite number of rational chemical modifications, as opposed to the countless number of essentially random chemical modifications that could be made, and of which any one might lead to a useful agonist or antagonist. Each chemical modification requires additional chemical steps, which while being reasonable for the synthesis of a finite number of compounds, quickly becomes overwhelming if all possible modifications needed to be synthesized. Thus through the use of the three-dimensional structure and computer modeling, a large number of these compounds can be rapidly screened on the computer monitor screen, and a few likely candidates can be determined without the laborious synthesis of untold numbers of compounds.
For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. Exemplary forcefields that are known in the art and can be used in such methods include, but are not limited to, the Constant Valence Force Field (CVFF), the AMBER force field and the CHARM force field.
The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
Alternatively, these methods can be used to identify improved modulating compounds from an already known modulating compound or odorant ligand. The composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified binding compounds or odorant ligands of improved specificity or activity.
Further examples of molecular modeling systems are the CHARMm and QUANTA programs (Polygen Corporation, Waltham, MA). CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modelling and analysis of molecular structure. QUANTA
allows interactive construction, modification, visualization, and analysis of the behaviour of molecules with each other.
Biosensors As used herein, the term "biosensor" means a sensor which converts an interaction between biomolecules into a signal such as an electric signal, so as to measure or detect a target substance. A biosensor of the invention can be any instrument which detects and/or identifies and/or quantifies odorants or similar volatile or non-volatile chemicals. A conventional biosensor is comprised of a receptor site for recognizing a chemical substance as a detection target and a transducer site for converting a physical change or chemical change generated at the site into an electric signal. Examples of biosensors incorporating receptor molecules are well known in the art and include those described in WO 00/70343.
Typically, a biosensor of the invention will comprise a polypeptide of the invention co-expressed with one or more accessory proteins such as a G protein, a sensory transduction mechanism, analogue to digital conversion, digital signal processing, pattern recognition, decision support and output.
In one embodiment, a fusion polypeptide of the invention comprises a resonance energy transfer (RET) acceptor and donor. RET is the non-radioactive transfer of energy from an excited state donor molecule to a ground state acceptor molecule. Energy transfer efficiency is dependent on the distance between the donor and acceptor, the extent of the spectral overlap and the relative orientation of the acceptor and donor dipoles. RET is increasingly being used to monitor inter and intra-molecular movements in biological systems. Examples of proteins which can be used include, but are not limited to, donors cyan fluorescent protein (CFP) and Renilla luciferase (RLuc) and their respective acceptors yellow fluorescent protein (YFP) and a variant of green fluorescent protein (GFP2). Fluorescent resonance energy transfer (FRET) has previously been used to quantify ligand binding by the a2-adrenergic receptor (Vilardaga et al., 2003; Hoffmann et al., 2005), whilst bioluminescent resonance energy transfer (BRET) has been used to monitor an intramolecular conformational change of 0-arrestin following GPCR activation (Charest et al., 2005).
As the skilled addressee will appreciate, similar receptor fusion proteins to those described in Vilardaga et al. (2003), Hoffmann et al. (2005), Charest et al.
(2005) and US 20060272037 can be used in biosensors of the present invention.
Insect Pests and the Control Thereof In a preferred embodiment, the insect pest is of the order Lepidoptera.
Examples include, but are not limited to, Achoroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmia feneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea, Helicoverpa armigera, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota stultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp., Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia hi, Udea rubigalis, Xylomyges curiails, and Yponomeuta padella.
The olfactory receptor genes studied as discussed herein may be used to identify compounds which interfere with the orientation and mating of a wide range of insects, especially Lepidopterans. Thus, the present invention enables the identification of compositions which disrupt insect mating by selective inhibition of specific receptor genes involved in mating attraction.
The identification of receptors for odorants is useful in developing new insect repellants and traps for the control of Lepidopterans and other insect pests.
The olfactory receptor genes studied using the materials, systems and methods of the present invention may be used to identify compounds which can be used as animal repellants.
The olfactory receptor genes studied using the materials, systems and methods of the present invention can also be used to identify compounds which attract specific insects to a particular location.
Aspects of the present invention are used in various methods which reduce or eliminate the levels of particular insect pests. Traps may also be utilized where trapped insects are killed by toxicant-containing poison baits where the insect may consume poisoned bait. The insect attractant compositions so identified may also be combined with an insecticide, for example as an insect bait in microencapsulated form. Alternatively, or in addition, the insect attractant composition may be placed inside an insect trap, or in the vicinity of the entrance to an insect trap.

EXAMPLES
Example 1 - Characterization of novel insect olfactory receptors Materials and Methods BmOr bioinformatics Known insect Ors whose sequences have been entered onto GenBank (National Center for Biotechnology Information) were used to search for similar genes in the silkworm genome sequence. Protein sequences were used to perform TBLASTN (Altschul et al., 1997) searches of assembled scaffolds available through two internet websites: http://kaikoblast.dna.affrc.go.jp/ (Silkworm Genome Research Program, National Institute of Agrobiological Sciences, Japan) and http://silkworm.genomics.org.cn/ (Beijing Genomics Institute, China). Genomic scaffold sequences were used to construct Or genes manually in the PAUP text editor (Swofford, 2001), using homology with known Or exons and an online program to predict exon/intron splice sites (SplicePredictor, http://deepc2.psi.iastate.edu/cgi-bin/sp.cgi). In some cases a single scaffold did not contain the complete Or gene;
where possible, 3' RACE was used to resolve the gene sequence (see below).
Divergent silkworm Ors were used in a second round of TBLASTN searches to find additional genes.

Phylogenetic analysis Conceptually translated protein sequences from silkworm Or genes identified in this study along with the 17 H. virescens Ors identified by Krieger et al., (2004) were used to construct a phylogenetic tree. The protein sequences were aligned using ClustaIX (Jeanmougin et al., 1998). Amino acid distances were calculated and multiple amino acid substitutions were corrected for using the maximum likelihood model, the BLOSUM62 amino acid exchange matrix, and uniform rates based on the actual sequences in TREE-PUZZLE v5.0 (Schmidt et al., 2002). Neighbor-joining followed by a heuristic search was employed to construct phylogenetic trees using PAUP* v4.0bl0 software (Swofford, 2001). Bootstrap analysis (n = 1000 replicates) was performed using uncorrected distances.

3' and 5' RACE
Total RNA was isolated from male and female adult moth antennae (n = 30 to 50 moths) using an RNeasy Mini Kit (Qiagen, Valencia, CA). Total RNA was quantified by absorption at a wavelength of 260 nm and its quality assessed on a 1%
agarose gel. For 3' RACE, lst strand cDNA was synthesized from 5 ug of total RNA
using a SuperScriptTM III First-Strand Synthesis System for RT-PCR kit (Invitrogen, Carlsbad, Ca USA) and a custom oligo dT primer, 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGT24-3' (SEQ ID
NO:83). First strand cDNA (20 uL final volume) was incubated with 4U of E.
coli Ribonuclease H (Invitrogen) for 20 min at 37 C. After heat inactivation at 65 C for 15 min, excess dNTPs and primer were removed using Microcon YM100 spin columns (Millipore Billerica, MA). Two uL of 1S` strand cDNA was used as template in a PCR reaction combining a gene-specific forward primer (BmOrs9, 10, 12, 14, 15, 19, 30, 33, 35, 37, 38, 42, 45 and 47) with a reverse primer specific to the polymerase promoter sequence (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-3' (SEQ ID
NO:84)) on the oligo dT primer used for lst strand cDNA synthesis. The stock PCR
mix contained: 1X Taq PCR buffer, 2 mM MgC12, 0.5 mM dNTPs, lU of Taq and 5 0.5U of Pfu polymerase per 25 uL (Stratagene, La Jolla, CA), and 0.4 uM
reverse primer. Two uL of 1 St cDNA and 1 uL of 10 uM gene specific primer (0.4 uM
final concentration) were added to 22 uL of lx PCR stock mix. PCR reactions were amplified using a Stratagene RoboCycler set at one cycle of 94 C for 2 min followed by 25 cycles of (94 C for 30 sec; 52 C for 30 sec; and 72 C for 2 min). Each PCR
10 tube was topped up with 25 uL of fresh lx PCR mix and 1 uL of 10 uM gene specific primer, amplified for a further 15 cycles (94 C for 30 sec; 52 C for 30 sec;
and 72 C
for 2 min), followed by a final cycle at 72 C for 7 min.
PCR reactions producing a product in the expected size range were gel purified using a MinElute Gel Extraction Kit (Qiagen, Valencia, CA) and sequenced using the 15 gene specific primer and the Taq Dye Deoxy Terminator Cycle Sequencing Kit combined with an automatic DNA sequencer (Applied Biosystems, Foster City, CA).
5' RACE was performed using the SMARTTM RACE cDNA Amplification Kit (Clontech, Mountain View, CA) following the manufacturers instructions.
Briefly, 50ng - lug of total antennal cDNA was primed at 70 C for 2 mins with 5' CDS
primer 20 and Smart II oligos (supplied in the kit). First strand cDNA was synthesised using PowerScriptTM Reverse Transcriptase (Clontech) at 42 C for 1.5hours. The 1St starnd cDNA was used as template in a PCR reaction with gene specific primers (reverse, BmOrs45 and 47) and the Clontech universal primer (forward) to produce gene specific PCR fragment that were sequenced.
Insects B. mori eggs and artificial diet were purchased from the Carolina Biological Supply Company (2700 York Road, Burlington, NC). The larvae were reared at 23 C
- 27 C on artificial diet, or white mulberry (Morus alba L.) leaves available locally during the summer season. Silkworm pupae were also provided as a gift from colonies reared by Dr. M. Goldsmith (University of Rhode Island, Kingston, RI). The antennae were dissected from male and female moths one to three days after emergence, frozen on dry ice, and stored at -80 C.

Quantitative real-time PCR
Total RNA (isolated from antennae collected from 30-50 female and 30-50 male moths) and lst strand cDNA were prepared as described for 3'RACE, with two exceptions: Genomic DNA was digested with DNAseI during on-column total RNA

purification (Qiagen, Valencia, CA USA) and a second time immediately before lst strand cDNA synthesis using the DNA-Free kit (Ambion, WoodwardAustin, TX), and, 15` strand cDNA was synthesized using the Invitrogen oligo dT(lg) primer.
PCR
primers were designed using ABI Primer Express 2.0 software (Applied Biosystems) set to select for an optimal primer annealing temperature of 59 C (58-60 C
range), amplicon sizes of 50-150 bp, a 3'GC clamp=0 and a minimum and maximum GC
content of 30 and 80%, respectively. In general, primers were designed using coding sequence close to the 3' end of the gene, and where possible, primers spanned an intron (Table 2). Real-time quantitative PCR was performed using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) and SYBR Green dye (SYBR Green PCR Master Mix, Applied Biosystems). The program began with a single cycle at 50 C for 2 min, followed by a single cycle at 95 C for 10 min and 40 cycles at (95 C for 15 sec; 60 C for 60 sec). Afterwards, the PCR products were heated to 95 C for 15 sec, cooled to 60 C for 15 sec and heated to 95 C for 15 sec to measure the dissociation curves.
Transcript levels of each Or gene were quantified from total RNA extracted from male and female adult antennae, and fifth instar larval abdomens, using the relative method (Relative Quantitation of Gene Expression, ABI PRISM 7700 Sequence Detection System, User Bulletin #2, Applied Biosystems ). The efficiency of each primer set was first validated by constructing a standard curve; a no-template control and six lOx serial dilutions of lst strand cDNA were prepared and the CT value (the cycle number at which the fluorescence intensity crosses the threshold line determined by the ABI Primer Express 2.0 software) of each Or gene calculated at each template dose (lx dose = 0.33 uL of lst strand cDNA). The CT value was plotted against the log(template dilution) and the slope and r 2 value of each regression line calculated. Expression of each Or gene in male and female antennae and larval abdomens was assessed at a template dose equal to 0.33 uL of lst strand cDNA
per well. A no-template control was included and all reactions were performed in triplicate. Dissociation curves were used to assess the purity of the PCR
reactions.
Or gene expression levels were calculated relative to the control gene B. mori ribosomal protein S3 (BmRPS3, Table 2) using the formula 2- CT (Livak and Schmittgen, 2001).

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After normalizing the CT values of each Or to the control gene BmRPS3, statistical differences between male and female antennae were tested by nested Analysis of Variance (ANOVA) using the SPSS for Windows Release 11Ø01 statistical package (SPSS inc., Chicago, IL). Technical replicates (n = 3 replicated wells on each qPCR plate) were nested within each biological replicate (n = 4 males and n = 4 females).

Results BmOr bioinformatics The sequences of seven silkworm Ors have been published. BmOrsl-3 named by Krieger et al., (2005) and Nakagawa et al., (2005) are synonymous. However, BmOrs 4-6 named by Krieger et al., (2005) are not synonymous with BmOrs 4-6 named by Nakagawa et al., (2005). BmOrs 4-6 as named by Nakagawa et al., (2005) are used herein since they were published first. The Ors referred to as BmOr4 and BmOr6 by Krieger et al., (2005) are synonymous with Nakagawa et al., (2005)'s BmOr5 and BmOr4 respectively. The Or referred to as BmOr5 by Krieger et al., (2005) shares 88% amino acid identity with Nakagawa et al., (2005)'s BmOr5 (their coding regions share 93% nucleotide identity), but since they are located on different genomic scaffolds, it is apparent they are different genes. Therefore, Krieger et al., (2005)'s BmOr5, which is a unique gene, is referred to herein as BmOr7 (J.
Krieger, Personal Communication).
Each new Or identified herein was named consecutively beginning with BmOr8 (see Table 2 for a summary, including GenBank accession numbers). The term chemoreceptor (Cr) is used to refer to the superfamily that includes the odorant and gustatory receptors (Ors and Grs).
Genomic scaffolds that encode unique Or exons were identified by conducting TBLASTN searches of the silkworm genome with known insect Or proteins (http://kaikoblast.dna.affrc.go.jp/, Silkworm Genome Research Program, National Institute of Agrobiological Sciences, Japan and http://silkworm.genomics.org.cn/, Beijing Genomics Institute, China) (Table 2). Or genes were annotated manually and assigned a new number beginning with BmOr8 (Table 2). In several cases the genomic scaffolds encoded only partial Or gene sequences due to their limited size and the presence of numerous intron sequences within the Or genes (BmOrs 45-47 in Figure IA for example). The N50 scaffold size (50% of the assembled genome sequence consists of scaffolds equal to or greater then the N50 value) of the silkworm genome is 26.9 kbp (Xia et al., 2004) compared to 990 kbp for the Tribolium casteneum genome for example (Human Genome Sequencing Center, Baylor College of Medicine, ftp://ftp.hgsc.bcm.tmc.edu/pub/data/Tcastaneum/Tcas2.0/README-Tribolium.txt). In some cases 3'and/or 5'RACE was used to amplify and sequence partial cDNA fragments in order to further annotate the gene structure. The coding regions and the translated protein sequences of BmOrs8-48 are presented in Supplemental Figure S2).
Although a complete assembly is not available, limiting the ability to assess Or clustering in the genome sequence, 14 Ors occur as seven tandem pairs on seven different scaffolds (BmOrs5 & 7, 19 & 20, 21 & 22, 23 & 24, 33 & 34, 38 & 39 and 46 & 48) providing evidence for some Or clustering. Tandem Ors share amino acid sequence similarity and group together on the phylogenetic tree (see below) suggesting they arose from recent gene duplication events. The nucleotide sequence of the open reading frame (ORF) of some paired Ors (e.g. BmOrs5 & 7 and 33 &
34) are almost identical (93 and 97% nucleotide identity, respectively) making it difficult to design unique primer sequences that also conform to the parameters required for real-time quantitative PCR.
The amino acid sequences of Ors from different insect orders have only low levels of identity, and tend to form order-specific lineages in phylogenetic trees (e.g.
Hill et al., 2002; Robertson and Wanner, 2006). However, there are some typical traits that can be used to support their designation as Ors. The last three intron/exon boundaries of many insect Or genes are spliced in the zero phase (between two codons rather then within a codon) a trait typical of the insect Ors in general (Robertson et al., 2003). The last exon is typically the smallest, and encodes some of the few highly conserved amino acid residues, particularly a Ser/Tyr/Ser motif (Figure 1B). A Ser residue (or in some cases a Thr residue) has been conserved in many insect Ors at the 7th codon position past the typical phase 0 splice site of the last intron/exon boundary. These features appear to have been broadly conserved in many (but not all) Or genes found in insect genomes sequenced to date (Robertson et al., 2003 and Robertson and Wanner, 2006). Examples include BmOrs19, 30, 45-47, AgOrl and DmOr46A in Figures IA and IB; these seven genes all have a phase 0 spliced intron/exon boundary located seven codon positions before the conserved Ser residue.

BmOr phylogenetics Including the seven unique Ors published by Krieger et al., (2005) and Nakagawa et al., (2005), the total number of silkworm Ors now identified is 48. A
neighbor-joining phylogenetic tree using corrected distances was constructed using 43 BmOrs (five partial sequences were excluded) and the 18 HvOrs that have been reported to date (Figure 2). Krieger et al., (2002) reported the sequences of chemoreceptors (Crs); 3 group together phylogenetically with the insect gustatory receptors (Grs), and 18 with the insect Ors (Robertson and Wanner, 2006), which are referred to herein as HvOrs for consistency.
Two results become evident from this analysis: first, most of the Or lineages are represented by both species, indicating diversification prior to the evolution of the ancestral bombycid and noctuid moths. All lineages with bootstrap support include representative Ors from B. mori, and even though fewer Ors have been identified from H. virescens (less then half compared to the silkworm), only three Or lineages lack HvOrs. Several Ors may be orthologous; examples include BmOrl3 & HvCr8, BmOrll & HvCr7, BmOrl4 & HvCr20, BmOrl8 & HvCr3, BmOr22 & HvCr19, BmOr24 & HvCrl2, BmOr25 & HvCr9, BmOr26 & HvCrl7, BmOr41 & HvCr10 (Figure 2).
Second, it is apparent that members of some of the lineages may share conserved functions (Figure 2). Seven B. mori and six H. virescens Ors group together in a single lineage that includes the pheromone receptors. All but two (BmOr9 and HvCr6) are expressed at higher levels in the male compared to female antennae (Table 3; Figure 3; Sakurai et al., 2004; Krieger et al., 2005;
Nakagawa et al., 2005). Furthermore, the pheromone receptors appear to form two main lineages, one that expanded in the bombycids and the other expanded in the noctuids. The bombykol receptor (BmOrl) may be an ortholog of HvCrl3, a receptor that is believed to detect the primary component of the H. virescens sex pheromone (Gohl and Krieger, 2006). Interestingly, this lineage has expanded in B. mori, while the lineage containing bombykal receptor (BmOr3) has expanded in H. virescens.
Similarly, BmOrs 45-48 appear to form a lineage of Ors whose expression is 6-8 times higher in female antennae (Figures 2 and 3, Table 3); whether this lineage also includes an orthorlogous Or from H. virescens remains to be determined.
The results of BLAST searches for each of the novel polypeptides performed when they were identified are provided in Table 4 where the closest known polypeptide at that time is indicated.

Table 3. Ratio of silkworm Or gene expression levels in female compared to male adult antennae determined by quantitative real-time PCR. Or gene expression levels in female and male antennae were calculated relative to the control gene BmRPS3 using the equation 2- CT (Livak and Schmittgen, 2001). Or expression values in 5 female antennae were divided by values in the male antennae and are reported as a female : male ratio. Ors whose expression was not detected in adult moth antennae (BmOr20, 21, 22, 25 and 42) are not included in the table.

BmOr No. female : male expression in anntennae 1 0.0001 2 0.9 3 0.0001 4 0.06 5&7 0.02 6 0.02 8 0.9 9 1.4 10 1.7 11 0.9 12 3.2 13 0.4 14 0.9 15 2.6 16 0.4 17 2.3 18 1.1 19 831.0 21 0.6 24 0.3 26 1.7 27 2.6 28 1.0 29 1.5 30 90.0 31 0.9 32 1.6 33&34 0.2 35 1.7 36 1.7 37 1.8 38 2.0 39 1.0 40 0.7 41 1.4 43 1.1 44 1.2 45 7.0 46 6.0 47 8.6 0 0 0 R ~ ~ ~ R
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Or gene expression in male and female antennae was normalized to the control gene BmRPS3 using the equation 2- CT (Livak and Schmittgen, 2001). BmRPS3 was consistently expressed at similar levels between the different tissues tested, indicating that the total RNA quantification was consistent. For example, CT values for BmRPS3 were 15.2, 16.7 and 16.6 in abdomen, female antennae and male antennae, respectively. Abdomen tissue from larvae was included as a control reference since it lacks any known olfactory sensilla (chemosensilla occur on the adult female ovipositor on the abdomen). CT values measured in abdominal tissue averaged 31.2 ( 1.1, 99% confidence interval) for all Ors tested, while values measured in antennal tissue ranged from 18 to 30. Or gene expression values in the abdomen tissue were consistently 10-4 times or more lower relative to BmRPS3 (Figure 3, minimum values rounded up to 10"4 for presentation). A no-template treatment included in each experiment controlled for template contamination. PCR products were analyzed by agarose gel electrophoresis to exclude contaminating genomic DNA as a source of template approximately half of the primer sets spanned an intron) and the purity of each PCR product was assessed by analyzing its dissociation curve. Or expression in the abdomen was typically 10-4 times that of BmRPS3, while Or expression in female and adult male antennae typically ranged from 10-3 to 1 times that of BmRPS3.
BmOr19 and BmOr3O were expressed abundantly in female (but not male) antennae at large female-biased ratios of 831x and 90x respectively (Table 3, Figure 3). This pattern of expression is opposite to that of the pheromone receptors BmOrl and BmOr3 that are expressed in male but not female antennae (Table 3; Figure 3).
BmOr19 is expressed at very high levels in female antennae, while BmOr3O is moderately abundant. In the phylogenetic tree (Figure 2) BmOr19 and 30 do not group together. BmOrs 8, 17, 20-22 and two H. virescens Ors (HvOrsl9 and 21) form a lineage with BmOrl9. Expression data is not available for HvCrsl9 and 21, and the other silkworm Ors found within this group were expressed at low levels or were not detected, with the exception of BmOrl7 (Figure 3). BmOrs14 and 33 & 34 (a tandem pair of Ors that share 97 % amino acid identity) along with two H. virescens Ors (HvOrl8 and 20) form a lineage with BmOr3O. BmOrs14 and 33/34 transcripts were detected in adult moth antennae and no sex-biased expression was observed;
expression data is not available for HvOrs 18 and 20.
Several Or transcripts were moderately more abundant in female compared to male antennae. In contrast to BmOrs 19 and 30, BmOrs 45-47 were expressed abundantly in both female and male antennae (46>45>47), but at ratios 6-8 times higher in female compared to male antennae (Table 3; Figure 3). BmOrs 45-48 all group together and may represent a lineage of Or genes with female biased expression (Figure 2). BmOr12 was also expressed at very high levels in both sexes, and was three times more abundant in female antennae. BmOrl2 groups together with two other silkworm receptors that are expressed at moderately high levels in the antennae, BmOrs 13 and BmOrl5 which expressed 2.6 times higher in female antennae (Table 3, Figure 2), and with H. virescens Or8.
The abundance of different Or transcripts relative to BmRPS3 varies over at least three orders of magnitude (Figure 3). Or gene transcripts that were 1000-fold less abundant relative to BmRPS3 are probably associated with a small proportion of the antennal sensilla. Conversely, abundant Or transcripts are likely expressed in commonly occurring olfactory neurons. For example, transcripts of the two pheromone receptors BmOrl & 3 were among the most abundant, their levels were equivalent to that of the control gene BmRPS3. Correspondingly, olfactory neurons tuned to the sex pheromone are associated with the most abundant class of sensilla on the male antennae (Heinbockel and Kaissling, 1996).
Real-time quantitative PCR accurately detected the sex-biased expression of five Or genes (BmOrl & 3-6) reported to be expressed at higher levels in male than female antennae (Table 3; Sakurai et al., 2004; Krieger et al., 2005). Of these, BmOrl and 3 transcripts were highly abundant in male but not female antennae (Table 3), consistent with their function as pheromone receptors. BmOrs 5 and 6 are the next most abundant male-biased Or genes and expression of each is approximately 50x higher in male antennae. BmOr 4 is expressed at moderate levels in both female and male antennae, yielding a moderate male-biased ratio of approximately 17x (Table 3).
The functions of BmOrs 4-6 have not been characterized. The primer sets used in this experiment did not distinguish between BmOrs 5 and 7, or between BmOrs 33 and 34.
The inventors assayed expression levels of the five male biased (BmOrs 1, 3, 4, 5/7, and 6) and five female biased (BmOrsl9, 30, 45, 46 and 47) silkworm Or genes in antennae collected from individual moths (n = 4 male and 4 female moths) (Figure 4). In each case, the results from this experiment using biological replication supported with statistical significance the male biased expression of BmOrs 1, 3, 4, 5/7, and 6, and the female biased expression of BmOrs19, 30, 45, 46 and 47, obtained in the previous experiments that used antennae from pooled individuals (Figures 2 and 5 4, Table 3). The levels of BmOr2 and BmOr9 were not statistically different between male and female antennae, also consistent with previous experiments where a significant sex-biased ratio was not observed (Table 3).

Discussion 10 The present inventors have identified 41 B. mori Ors bringing the total number to 48. The constructed B. mori genes were assigned to the Or family based upon amino acid similarity with known insects Ors and characteristic gene structure and amino acid motifs. Supporting their annotation, the majority of the putative Or genes were expressed in the antennae of adult moths. Several of the Or genes may be 15 expressed exclusively in the larval sensory organs, as is the case in D.
melanogaster (Fishilevich et al., 2005), explaining the failure to detect them in adult antennae (BmOrs2O, 22, 23, 25 and 42).
In phylogenetic analyses, insect odorant receptors generally form order-specific branches reflecting rapid rates of evolution within the gene family (Robertson 20 and Wanner, 2006). This pattern is also observed between distantly related groups within the same order, such as the mosquitoes and flies, one of the earliest taxonomic divisions within the Diptera (Hill et al., 2002). This high degree of divergence in the amino acid sequences exhibited by the Ors makes it very difficult to use homology-based discovery approaches to identify the Ors from insects whose genomes have not 25 been sequenced.
Regardless, the silkworm Or genes identified herein can be used as probes to screen cDNA libraries constructed from the antennae of important pest species found in other taxonomic families such as the Noctuidae. Homology-based approaches will be particularly useful to identify Ors that mediate important pest behaviors such as 30 host selection, feeding and oviposition.
BmOrl & 3 are not expressed at significant levels in female antennae (Figure 3; Sakurai et al., 2004; Krieger et al., 2005). Absence of a receptor from the antennae of one sex (as opposed to expression in both sexes at a biased ratio) suggests that it may mediate an olfactory behavior entirely specific to that sex, as is the case with the 35 silkworm sex pheromones. BmOr6 on the other hand is expressed in both male and female antennae, but 50 times more abundantly in male antennae (Table 3), suggesting that the odor(s) that its detects may mediate behaviors more prominent in male moths (such as unidentified female pheromones or conspecific odors). Some H.

virescens sensory neurons on the female antennae respond to one of the six female produced pheromone components (Hillier et al., 2006), a scenario that could explain expression of the receptor in both sexes but at greater levels in male antennae.
Similarly, several silkworm Or genes are expressed at moderate to large female-biased ratios. BmOrs45-47 are expressed abundantly in the antennae of both sexes, but six to eight times higher in the females. This dimorphism may reflect an enhanced sensitivity of female antennae to specific odors used for host plant discrimination. Interestingly, BmOr19 and BmOr3O are expressed at large female-biased ratios (approximately 800 and 90 times, respectively). BmOr19 is likely expressed in a high proportion of olfactory neurons on the female antennae based upon the high levels of its transcripts in female antennae.
Comparatively, BmOr3O is less abundant and therefore likely expressed in a smaller proportion of sensilla on the female antennae. Based on their low levels of expression in male antennae, BmOrl9 and BmOr30 may mediate the detection of female-specific olfactory cues that mediate female-specific behaviors. These results are consistent with the existence of two sexually isomorphic glomeruli on the antennal lobes of adult B. mori females (Koontz and Schneider, 1987). Similarly, the antennal lobes of adult M. sexta females also have two enlarged glomeruli, one of which responds to linalool (King et al., 2000; Rospars and Hildebrand, 2000;
Reisenman et al., 2004), a fact that may indicate the conservation of specific olfactory pathways in female moths. Koontz and Schneider (1987) suggested that such pathways may function in the detection of odors related to host plant selection for feeding and oviposition or for the detection of male produced sex pheromones. As many as 66%
of the sensilla tested on female H. virescens antennae responded to conspecific chemical cues, including pheromones produced by the male that influence mating behavior (Hillier et al., 2006).

Example 2 - Analysis of receptor-ligand binding Functional analysis was performed using a calcium imaging assay which has been described by Kiely et al., (2007). Briefly Spodoptera frugiperda (Sfl9) cells were transiently transfected with a pIB vector using Escort IV (Sigma). Transfected cells were incubated for 48hours to allow the expression of olfactory receptor before calcium imaging of responses to ligands were assessed. Fluo4 was used as a calcium indicator and fluorescence images were recorded using a Leitz digital still camera.
Images were recorded every 10 seconds for 50 seconds after the addition of;
saline (as a control), the test ligand and lonomycin (to determine maximal fluorescence).
Images were analysed using Metafluor imaging system and AF was calculated for given concentrations. AF is determined as the ratio of change in fluorescence from = 57 basal levels after the addition of a ligand to maximum change in fluorescence from basal levels after the addition of lonomycin.
EC50 curves were created in Graphprism with all points based on the average AF of at least 4 individual cells.
Following the same methodology described for the expression and characterization of EposOr3 (PCT/AU2007/000510), BmOr19, 30, 45 and 47 were screened for detection of 25 different odours (Table 5) representing a diverse range of chemical properties. Each of these molecules are plant volatiles and/or components of insect pheromones, and hence uncoupling of signalling will effect insect behaviour.
Functional assays were carried out at final concentrations of 10"6 and 10"$
for each odour.

Table 5. Odours tested in calcium aging in assays a-pinene Citral Limonene a-terpineol Ethyl-benzoate Linalool p-cresol Ethyl-butyrate Methyl benzoate 1,4-cineole Ethyl-hexanoate Methyl salicylate 2-phenyl-ethanol Eugenol Myrcene Benzaldehyde Farnesene Octen-3-ol Benzoic acid Geraniol Trans-2-hexanal Butanal Geranyl acetate Caryophyllene Hexanol Linalool was the only ligand detected that produced a response from BmOrl9 (Table 6, Figure 5).

Table 6. EC50 values for B. mori Or odours.
Receptor Odour EC50 BmOr19 Linalool (4.69 1.57) x 10"9 M
BmOr45 Benzoic acid (1.44 1.36) x 10-10M
2-phenyl-ethanol (8.89 2.59) x 10-9 M
Benzaldehyde (5.86 1.67) x 10-9 M
BmOr47 Benzoic acid (1.42 1.52) x 10""M

BmOr45 responded most highly to 2-phenylethanol, benzoic acid and benzaldehyde (Table 6, Figures 6 to 8). Smaller responses were seen for ethyl benzoate and methyl benzoate although these were inconsistent and EC50 data was not obtained.

BmOr47 responded highly to benzoic acid (Table 6, Figure 9) and only showed very weak responses to high concentrations of 2-phenyl ethanol and benzaldehyde.
BmOr3O did not respond to any of the tested ligands.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
All publications discussed and/or referenced herein are incorporated herein in their entirety.
This application claims priority from US 60/971,133, the entire contents of which are incorporated herein by reference.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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Claims (56)

1. A substantially purified and/or recombinant polypeptide comprising amino acids having a sequence as provided in any one of SEQ ID NOs: 1 to 41, a biologically active fragment of any one thereof, or an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs: 1 to 41, wherein the polypeptide is an olfactory receptor.

2. A substantially purified and/or recombinant polypeptide comprising amino acids having a sequence as provided in any one of SEQ ID NOs: 12, 23, 38, 39, and 41, a biologically active fragment of any one thereof, or an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs: 12, 23, 38, 39, 40 and 41, wherein the polypeptide is a female specific olfactory receptor.

3. The polypeptide of claim 2 which comprises an amino acid sequence which is at least 90% identical to any one or more of SEQ ID NOs: 12, 23, 38, 39, 40 and 41.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide can be purified from a Lepidopteran.

5. The polypeptide according to any one of claims 1 to 4 which is fused to at least one other polypeptide.

6. An isolated and/or exogenous polynucleotide comprising nucleotides having a sequence as provided in or complementary to any one of SEQ ID NOs: 42 to 82, a sequence which is at least 50% identical to any one or more of SEQ ID NOs: 42 to 82, a sequence which hybridizes to any one or more of SEQ ID NOs: 42 to 82, or a sequence which encodes a polypeptide according to any one of claims 1 to 5.

7. The polynucleotide of claim 6 which encodes an olfactory receptor.

8. The polynucleotide of claim 6 which encodes a female specific olfactory receptor.

9. An oligonucleotide which comprises at least 19 contiguous nucleotides of a polynucleotide according to any one of claims 6 to 8.

10. An isolated and/or exogenous polynucleotide which, when present in a cell of an insect, interferes with chemosensory perception of the insect when compared to a cell of an insect that lacks said polynucleotide, wherein the polynucleotide comprises nucleotides having a sequence as provided in or complementary to any one of SEQ ID
NOs: 42 to 82, a sequence which is at least 50% identical to any one or more of SEQ
ID NOs: 42 to 82, a sequence which hybridizes to any one or more of SEQ ID
NOs:
42 to 82, a sequence which encodes a polypeptide according to any one of claims 1 to 5, and/or which comprises an oligonucleotide of claim 9.

11. The polynucleotide of claim 10, wherein the polynucleotide is selected from:
an antisense polynucleotide, a catalytic polynucleotide and a double stranded RNA.

12. The polynucleotide of claim 11 which is a catalytic polynucleotide capable of cleaving a polynucleotide according to any one of claims 6 to 8.

13. The polynucleotide of claim 11, which is a double stranded RNA (dsRNA) molecule comprising an oligonucleotide according to claim 9, wherein the portion of the molecule that is double stranded is at least 19 basepairs in length and comprises said oligonucleotide.

14. A vector comprising or encoding the polynucleotide according to any one of claims 6 to 8 or 10 to 13.

15. The vector of claim 14, wherein the polynucleotide, or sequence encoding the polynucleotide, is operably linked to a promoter.

16. A host cell comprising at least one polynucleotide according to any one of claims 6 to 8 or 10 to 13, and/or at least one vector of claim 9 or claim 10.

17. A process for preparing a polypeptide according to any one of claims 1 to 5, the process comprising cultivating a host cell according to claim 16 encoding said polypeptide, or a vector of claim 15 encoding said polypeptide, under conditions which allow expression of the polynucleotide encoding the polypeptide, and recovering the expressed polypeptide.

18. An antibody which specifically binds a polypeptide according to any one of claims 1 to 4.

19. A composition comprising a polypeptide according to any one of claims 1 to 5, a polynucleotide according to any one of claims 6 to 8 or 10 to 13, a vector of claim 14 or claim 15, a host of claim 16 and/or an antibody of claim 18, and one or more acceptable carriers.

20. A kit comprising a polypeptide according to any one of claims 1 to 6, a polynucleotide according to any one of claims 6 to 8 or 10 to 13, a vector of claim 14 or claim 15, a host of claim 16, an antibody of claim 18, and/or a composition of claim 19.

21. A method of identifying a molecule that binds to a polypeptide according to any one of claims 1 to 4, the method comprising:
i) contacting a polypeptide according to any one of claims 1 to 5 with a candidate compound, ii) determining whether the compound binds the polypeptide.

22. A method of identifying a molecule that binds to a polypeptide according to any one of claims 1 to 4, the method comprising:
a) exposing a polypeptide according to any one of claims 1 to 5 to a binding partner which binds the polypeptide, and a candidate agent, and b) assessing the ability of the candidate agent to compete with the binding partner for binding to the polypeptide.

23. The method of claim 22, wherein the binding partner is detectably labelled.

24. A method of identifying a molecule that binds to a polypeptide according to any one of claims 1 to 4, the method comprising:
i) contacting a protein complex comprising a polypeptide according to any one of claims 1 to 5 with a candidate compound, ii) determining whether the compound binds the complex.

25. The method according to any one of claims 21 to 24, wherein the polypeptide is expressed in a cell.

26. The method of claim 25, wherein the polypeptide spans the cell membrane.

27. The method of claim 25 or claim 26, wherein the cell is an insect cell.

28. The method of claim 27, wherein the insect cell is an olfactory receptor neuron.

29. The method of claim 27 or claim 28, wherein the insect cell is a Lepidopteran cell.

30. A method of identifying a molecule that modulates the activity of a polypeptide according to any one of claims 1 to 4, the method comprising:
i) contacting a cell comprising a polypeptide according to any one of claims 1 to 5 with a candidate compound, ii) determining whether the compound modulates a physiologic activity of the cell.

31. A method of identifying a molecule that modulates the activity of a polypeptide according to any one of claims 1 to 4, the method comprising:
i) contacting a first cell comprising a polypeptide according to any one of claims 1 to 5 with a candidate compound, ii) contacting a second cell lacking the polypeptide with the candidate compound, and iii) determining whether the compound modulates a physiologic activity in the first or second cell, wherein the first and second cell are the same cell type, and wherein a compound that modulates a physiologic activity in the first cell but not the second cell is a modulator of the polypeptide.

32. The method of claim 31, wherein the cell is a cell of an organism.

33. The method of claim 32, wherein the first cell and second cell are cells of the same cell type from two different individuals of an organism of the same species.

34. The method of claim 32 or claim 33, wherein the organism is a Lepidopteran.

35. The method according to any one of claims 30 to 34, wherein the physiologic activity is determined by analysing a behavioural activity of the organism.

36. The method according to any one of claims 30 to 35, wherein the physiologic activity is G-protein activity.

37. The method of claim 36, wherein G-protein activity is determined by measuring calcium ion and/or cyclic AMP concentration in the cell.

38. The method according to any one of claims 30 to 35, wherein the physiologic activity is determined using an electroolfactogram.

39. A method of screening for a compound that modulates the activity of a polypeptide according to any one of claims 1 to 4, the method comprising using the structural coordinates of a crystal of the polypeptide to computationally evaluate a candidate compound for its ability to bind to the polypeptide.

40. The method according to any one of claims 21 to 39, wherein the compound is an odorant.

41. The method according to any one of claims 30 to 40, wherein the compound is an antagonist of the physiologic activity.

42. The method according to any one of claims 30 to 40, wherein the compound is an agonist of the physiologic activity.

43. A compound identified using a method according to any one of claims 21 to 42.

44. A method for controlling an insect pest, the method comprising exposing the insect pest to an antagonist of claim 41.

45. A method for controlling an insect pest, the method comprising exposing the insect pest to an agonist of claim 42.

46. A biosensor comprising a polypeptide according to any one of claims 1 to 5.

47. A transgenic non-human animal comprising an exogenous polynucleotide, the polynucleotide encoding at least one polypeptide according to any one of claims 1 to 5.

48. A transgenic non-human animal comprising an exogenous polynucleotide according to any one of claims 6 to 8 or 10 to 13, and/or a polynucleotide encoding therefor.

49. A transgenic plant comprising an exogenous polynucleotide, the polynucleotide encoding at least one polypeptide according to any one of claims 1 to 5.

50. A transgenic plant comprising an exogenous polynucleotide according to any one of claims 10 to 13, and/or a polynucleotide encoding therefor.

51. A method for controlling an insect pest, the method comprising delivering to the insect a polynucleotide according to any one of claims 10 to 13, and/or a polynucleotide encoding therefor.

52. The method of claim 51, wherein the polynucleotide is delivered by exposing the insect to a transgenic plant according to claim 50, wherein the insect eats the plant.

53. A method of controlling female insect pests, the method comprising exposing the female insect pests to a ligand which binds a polypeptide comprising amino acids having a sequence as provided in any one of SEQ ID NOs: 12, 23, 38, 39, 40 and 41, a biologically active fragment of any one thereof, or an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs: 12, 23, 38, 39, 40 and 41.

54. The method of claim 53, wherein the ligand disrupts mating.

55. The method of claim 53 or claim 54, wherein the ligand is linalool, 2-phenylethanol, benzoic acid, benzaldehyde, ethyl benzoate or methyl benzoate.

56. The steps, features, integers, compositions and/or compounds disclosed herein or indicated in the specification of this application individually or collectively, and any and all combinations of two or more of said steps or features.