CA2563886A1 - Compositions and methods for controlling insects related to the octopamine receptor - Google Patents

Abstract

A screening method for identifying compounds that are effective insect control agents includes providing cells expressing an octopamine receptor, adding the compounds to the cells, and measuring the effects of the compounds and compositions. The effects of the compounds may be determined by measuring the binding affinity of the compounds to the octopamine receptor or measuring the change in intracellular cAMP or Ca2+ levels.

Description

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COMPOSITIONS AND METHODS FOR CONTROLLING INSECTS
RELATED TO THE OCTOPAMINE RECEPTOR
FIELD OF THE INVENTION
The present invention relates to compounds, compositions and method for controlling insects.
BACKGROUND OF THE INVENTION
Animals have chemosensory and mechanosensory systems that recognize a large array of environmental stimuli, generating behavioral responses. Many behavioral studies have been conducted to understand the genetics of these systems. The olfactory system plays a crucial role in the survival and maintenance of species, particularly in insects.
Biogenic amines serve a neurotransmitter or neuromodulator role in the olfactory system.
The biogenic amine, octopamine, has a prominent role in insects and other invertebrates as it is involved in the regulation of multiple physiological events, for example, effects on muscular systems, sensory organs, endocrine tissues as well as learning and behavior, Octopamine (OA) occurs in large amounts in the nervous systems of species representing the phylum Arthropoda, including the classes Insecta and Crustacea. OA has a broad spectrum of biological roles in insects acting as a neurotransmitter, neurohormone and neuromodulator. OA
exerts its effects through interaction with at least four classes of membrane bound receptors that belong to the family of G-protein coupled receptors (GPCRs). All members of GPGRs share the C01111no11 lllOtlf of seven transmembrane (TM) domains.
When a GPCR is activated, depending on its type and the protein to which it binds, changes in intracellular concentrations of cAMP, Ca'~ or both often take place. Since changes in intracellular levels of CAMP or Ca'+ are the most commonly found cellular responses to biogenic amine treatments (e.g., serotonin, dopamine, octopamine, etc.), they are used to functionally classify receptor subtypes. As a result of GPCR activation, intracellular CAMP
levels can either be elevated or reduced. The cellular response strictly relies on the specificity of interaction between the receptor and the G protein (See e.~., Gudermann T, I~allcbrenner F, Schultz G . 1996, "Diversity and selectivity of receptor-G protein interaction," Annu Rey Pharmacol Toxicv 1 36:
429-459; and Gudermann T, Schoneberg T, Schultz G. 1997, "Functional and structural complexity of signal transduction via G-protein-coupled receptors," Annu Rev Neurosci 2 0: 399-427, both of which are incorporated herein by this reference). When the receptor binds to Gs-type protein, the activated Gas subunit will interact with adenylyl cyclase (AC) in the plasma membrane. This leads to an increase of AC activity and production of CAMP from ATP.
Several biogenic amine receptors are also known to inhibit AC activity. This effect is mediated by interaction of the receptor with inhibitory G protein (Gi).
Interaction of AC wvith activated Gai subunits most likely competes witlybinding of activated Gas subunits and t(~ ereby interferes with AC activation.
Another pathway that is activated by several biogenic amine receptors results in a rise of intracellular Ca2+ levels. In such a scenario the amine-activated receptor binds to G protein's of the Gq/o family (See e.g., supya, Gudermann et al., 1996 and Gudermann et al., 1997). TI~ a activated Gaq/o subunits bind to and stimulate phospholipase C (PLC) activity.
The enzyme hydrolyzes a membrane-bound substrate, phosphatidylinositol 4,5-bisphosphate which giv es rise to two second messengers IP3 and DAG. After binding of IP3 to its receptors, the calciwn channel pore is opened and Ca2+ is released into the cytoplasm. Ca''+ ions play a vital role in the regulation of many cellular functions by binding to members of large family of Ca'+-bindimg proteins and/or directly controlling enzymatic or ion channel activities.
Multiple insect species have been utilized to understand the biological functions amd pharmacological characteristics of octopamine receptors. Studies with Peripdaneta afne~i~ana (American cockroach) have provided insight into the pharmacology and second messenger signaling of octopamine through octopamine receptors. For example, octopamine has been found to activate adenylate cyclase in certain cells in this species. Furthermore, octopamine has been found to increase inositol triphosphates in certain cells in this species.
As the octopaminergic system is believed to be unique to invertebrate physiology, this pathway has been proposed to offer a target for invertebrate pesticides with potential for low vertebrate toxicity, Formamidine-like chemicals have been found to be octopaminergic agonists and inhibit the uptake of sodium-sensitive octopamine in cel-tain insects; for example, the formamidine pesticides chlordimefonn and demethylchloridimeform were found to target the octopamine signaling pathway in cel-tain invertebrates, including Pe~~iplar7eta an~enicaJZa, To provide insight into the design of octopamine agonists that could be used as potential insecticides, structure function analyses have been performed with 2-(arylimino)oxazolidines and 2-(substituted benzylamino)-2-oxazolines in regard to activation of the octopamine sensitive adenylate cyclase in certain cells in Peg°iPlaf~eta Aroe~ica~a. More recently, it has been suggested that one site of action for the insecticidal activity of plant essential oils against Per~ilalaneta anze~~icaf~a is the octopaminergic system and that octopamine receptors may be targeted by these compounds, as described in Enan, E., 2001, "Insecticidal activity of essential oils: octopaminergic sites of action," Comp. Biochem. Physiol. C Toxicol. Pharmacol. 130, 325-327, which is incorporated herein by this reference.
Identifying plant essential oils and combinations thereof, having 111SeCt-COlltl'Olllllg activity is particularly desirable given that many such compounds do not produce unwanted or harmful affects on humans, other animal species, and certain plants. However, identifying the most effective plant essential oils and combinations thereof requires random selection and use of tedious screening methods, which, given the vast number of plant essential oils and possible combinations thereof, is a substantially impossible task.

As such, there is a need in the art for an improved method for screening compounds and compositions for insect control activity.

SUMMARY OF THE PRESENT INVENTION
The present invention addresses the above identified problems, and others, by providing a screening method for identifying compounds and compositions that are effective insect control agents; a screening method for identifying compounds and compositions that are effective species-specific insect control agents; compounds and compositions isolated from the screening methods; cell lines expressing an octopamine receptor; and isolated nucleic acid molecule sequences.
DESCRIPTION OF THE DRAWINGS
Figure lA is an alignment of the nucleic acid sequence and the translated amino acid sequence from Pa oar, of SEQ ID NO: 1 and SEQ ID NO: 2;
Figure 1B is the nucleic acid sequence fr0111 Pa oal of SEQ ID NO: 1, with the seven putative transmembrane domains (TM) overlined and numbered 1 through 7, the stop codons (SC) underlined, and the initiation codon (M) underlined;
Figure 2 is an alignment of the translated amino acid sequences of Pa oar of SEQ ID NO: 2 and OAMB of SEQ ID NO: 3, with the seven putative transmembrane domains (TM) overlined and numbered 1 through 7;
Figure 3A is saturation binding curve of'H-yohimbine to Pa oar, where total binding is designated by the squares, nonspecific binding is designated by the triangle, and specific binding is designated by the inverted triangle;
Figure 3B is saturation binding curve of 3H-yohimbine to OAMB, where total binding is designated by the squares, nonspecific binding is designated by the triangle, and specific binding is designated by the inverted triangle;
Figure 4 is a hydropathy profile of Pa oar with the transmembrane domains (TM) numbered 1 through 7;

Figure 5 depicts the similarity between octopamine and tyramine receptors -lrom different insect species;
Figure 6 is a graph depicting the change of intracellular cAMP levels in HEK-293 cells expressing Pa oal in response to treatment with various concentrations of either octopamine (OA) or tyramine (TA); and Figures 7 is a graph depicting the change in intracellular calcium levels in HEK-293 cells expressing Pa oal in response to treatment with either 100 nM octopamine (OA) or 100 nM
tyramine (TA);
Figure 8 is a bar graph depicting the change in intracellular CAMP levels in expressing Pa oal in response to treatment with 0, 100 nM, or 1 EIM octopamine (OA) in the presence and absence of 20 L~M BAPTA/AM, a calcium chelator;
Figure 9 is a bar graph depicting the cAMP response to octopamine through Pa oai and OAMB expressed in HEK-293 cells where the cells expressing either receptor are treated with 10 p,M octopamine and the level of CAMP is determined;
Figures 10A and lOB are graphs depicting the calcium response to octopamine through Pa oal and OAMB, respectively, expressed in HEIR-293 cells;
Figure 11 is a depiction of the chemical structures ofp-cymene [methyl( 1-methylethyl)benzene], eugenol [2-methoxy-4-(2-propenyl)phenol], traps-anethole [ I -methoxy-4-(I-propenyl)benzene], cinnamic alcohol [3-phenyl-2-propen-1-of], a-terpineol [p-menth-1-en-8-0l], methyl salicylate [2-hydroxybenzoic acid methyl ester], 2-phenylethyl propionate, and geraniol [3,7-dimethyl-2,6-octadien-1-of];
Figure 12 is a bar graph depicting the efi°ect of certain plant essential oils on speci lic bllldlllg of 3H-yohimbine to Pa oai and OAMB;
Figure 13 is a bar graph depicting the effect of certain plant essential oils on cAMP levels in HEK-293 cells expressing either Pa oal or OAMB; and Figures 14A-14F are graphs depicting the effect of certain plant essential oils on intracellular calcium [Ca2+]; levels in HEK-293 cells either transfected with Pa oa, or OAMB.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes: a screening method for identifying compounds and compositions that are effective insect control agents; a screening method for identifying compounds and compositions that are effective species-specific insect control agents; compounds and compositions isolated from the screening methods; transfected cell lines;
alld isolated IluclelC
acid molecule sequences.
The present invention includes: an isolated nucleic acid molecule sequence which encodes a protein that binds a biogenic amine, resulting in changes in intracellular concentrations of CAMP, Ca'+, or both, having a nucleotide sequence.of SEQ ID NO: l, or a fragment or derivative thereof andJor having an amino acid sequence of SEQ ID NO: 2, or a fragment or derivative thereof; an isolated nucleic acid molecule of having at least about 30°fo similarity to the nucleotide sequence of SEQ ID NO: 1, wherein the isolated nucleic acid molecule encodes a protein, resulting in changes in intracellular concentrations of cAMP, Ca2+, or both;
an isolated nucleic acid molecule of having at least about 30% similarity to the nucleotide sequence of SEQ ID NO:
l, wherein the molecule encodes an octopamine receptor or a protein having an amino acid sequence of SEQ ID NO: 2, or a fragment or derivative thereof; and an isolated nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1, or a fragment or derivative thereof, wherein the molecule encades a protein designated Pa oal. SEQ ID NO: 1 and SEQ
ID NO: 2 are shown in alignment in Figure 1A and SEQ ID NO: 1 is also provided in Figure 1 B. Fragments and derivatives of the sequences shall include transmembrane domains (TM) 3, 5 and 6.
Fragments and derivatives of the sequences may exclude, for example, portions upstream of TM
1, portions upstream of TM 2, or portions downstream of TM 7.

The present invention also includes: a strain of cells including a DNA vector having a nucleic acid sequence of SEQ ID NO:1; a strain of cells expressing an octopamine receptor cloned from an insect species of interest; a strain of cells expressing an octopamine receptor cloned from Pe~ipla~reta Anae~~icaha (Pa oar ); a strain of cells expressing a protein having an amino acid sequence of SEQ ID NO: 2, or fragments or derivatives thereof, wherein the fragments or derivatives thereof bind octopamine; a strain of cells expressing an octopamine receptor cloned from Drosoplzila n~elay7ogaste~~ (OAMB); a strain of cells expressing a protein having an amino acid sequence of SEQ ID NO: 3, or fragments or derivatives thereof, wherein the fragments or derivatives thereof bind octopamine. The transfected cells may be mammalian cells or insect cells; for example, they may be African green monkey kidney COS-7 cells (COS-7 cells) or human embryonic kidney-293 cells (HEIR-293 cells).
The present invention also includes a screening method of using a cell line expressing an octopamine receptor to identify compounds and compositions that are effective insect control agents. .For example, the octopamine receptor expressed by the cell line may be Pa oal; or have an amino acid sequence of SEQ ID NO: 2, or fragments or derivatives thereof, wherein the fragments or derivatives thereof bind octopamine.
The present invention also includes a method of using multiple cell lines, wherein the cell lines are transfected with octopamine receptors from different insect species of interest, to identify compounds and compositions that are effective species-specific insect control agents. For example, a cell line expressing Pa oal and a cell line expressing OAMB could be used to screen compounds and compositions having insect control activity which is specific to Peg°i~laneta Anzef°ica~ra or to DT~os~plzila n~elahogaste~~.
The present invention also includes compounds and compositions having the ability to control target insects, which compounds and/or compositions are identified using the screening methods of the present invention. These compounds and/or compositions may include compounds that are general regarded as safe (GRAS compounds) meaning that they do not produce unwanted or harmful affects on humans and other non-target animal species and that they are exempt from the Environmental Protection Agency's (EPA) pesticide registration requirements. The compounds and/or compositions of the present invention include certain plant essential oils identified using the screening methods of the present invention.
The CO111pOLl1ldS alld CO111pOS1tIO11S Of the pl'e5e11t I11Ve11t1011 COlltr0l 111S2CtS by targeting aI7 octopamine receptor, resulting in a disruptive change in the intracellular levels of cAMP, Ca2+ or both. For purposes of simplicity, the term insect has been and shall be used through out this application; however, it should be understood that the term insect refers, not only to insects, but also to arachnids, larvae, and like invertebrates. Also for purposes of this application, the term "insect control" shall refer to repelling or killing an insect.
The present invention is fu l-ther illustrated by the following specific but non-limiting examples.

PREPARATION OF STABLYTRANSFECTED COS-7 CELL LINES and HEK-293 CELL LINES WITH OCTOPAMINE RECEPTOR
A. ISOLATION OF A cDNA ENCODING A G-PROTEIN-COiJPLED RECEPTOR
FROM PERIPLANETA AMERICANA
G protein-coupled receptors from insects and a tick that are demonstrated to be octopamine receptors or have significant DNA similarity to known octopamine receptors are aligned using the program DNAStar (Ma). The following degenerate oligonucleotides are designed based on this alignment: Transmembrane (TM) VI oligonucleotide 5'TACAAGCTTTG(C, T~TGG(C, T) (G, T) (A, C, G, T)CC(A, C, G,T)TTCTT3' (SEQ ID
NO:
4), and TM VII oligonucleotide 5'CATGCGGCCGCTTT(A, C, G, T) (A, CAA, C) (A, G)TA(A, C, G T)CC(A, C)AGCCA3' (SEQ ID NO: 5), The underlined sequence corresponds to the TM
regions.
The TM VI oligonucleotide contains a Hinc~lll site and the TM VII
oligonucleotide contains a Notl site flanking the TM sequences. Total RNA from the heads of mixed sex adult American cocla~oaches that have the antennae excised is prepared by ultracentrifugation through cesium chloride, as described in Chirgwin et al., 18 Biochemistry 5294-5299 (1979), and is reverse transcribed into cDNA using random hexamers and murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA), The polymerase chain reaction (PCR) is performed on this cDNA using AmpliTaq polymerase (Applied Biosystems) and the TM VI and VII oligonucleotides at final concentrations of about 5 pM, The reaction conditions are about 95 ~C, about 5 min for about one cycle; about 95 UC, about 45 s, about 40 ~C, about 2 min, about 72 ~C, about 30 s for about three cycles; about 95 ~C, about 45 s, about 55 ~C, about 2 171111; about 72 ~C, about 30 s for about 37 cycles; and about 72 ~C, about 10111111 for about one cycle, Products are digested with Hi~rdlll and Notl and ligated into pBI~-RSV
(Stratagene, La Jolla, CA). Inserts are sequenced and compared to 1C110W11 genes by Seal'Chlllg the NCBI database with the Blast program.
To obtain the corresponding cDNA for an approximately 101 nucleotide fragment with the highest similarity to octopamine receptors from other species, 5' and 3' rapid amplification of cDNA ends (RACE) are performed using the SMART RACE cDNA amplification system (Clontech, Palo Alto, CA), Poly(A) RNA is prepared from total RNA isolated from the head of Pei~ipla~eta amerieana using an oligo-dT column as per the manufact~.irer's protocol (A111e1'S11a111 Biosciences, Piscataway, NJ). The poly(A) RNA is used as template in the RACE
reverse transcription reaction for production of 5' and 3' RACE cDNA as per the manufacturer's instructions. The gene specific oligonucleotides used for the RACE PCR are 5' RACE
oligonucleotide 5'CAGTAGCCCAGCCAGAAGAGGACGGAGAAG3' (SEQ ID NO: 6), and 3' RACE oligonucleotide 5'GCTGGCTGCCGTTC'TTCACCATGTACCTGG3' (SEQ ID NC): 7), 5' RACE and 3' RACE polymerase chain reactions are each about 50 y1 and consist of about 2.5 Etl of the respective cDNA reaction, about 0.2 ~.M of the gene specific oligonucleotide and the additional RACE components including Advantage 2 polymerase as per the manufacturer (Clontech). The cycling conditions for the 5' RACE are about 95 ~C, about 1 min for about one cycle; about 94 ~C, about 20 s, about 72 ~C, about 3 min for about five cycles; about 94 ~C, about s, about 70 ~C, about 10 s, about 72 ~C, about 3 111111 for about five cycles;
about 94 ~C, about 20 20 s, about 68 ~C, about 10 s, about 72 ~C, about 3 niin for about 32 cycles; and about 72 ~C, about 10 min for about one cycle.
An approximately 1.9 lcb product is gel purified and further, amplified using the same oligonucleotides, Advantage 2 polymerase and cycling parameters of about 95 ~C, about 3 min for about one cycle; about 94 ~C, about 20 s, about 68 ~C, about 10 s? about 72 nC, about 3 min for about 35 cycles; and about 72 ~C, about 10 min for about one cycle. To facilitate T/A ligation, the product is A-tailed by precipitating with ethanol, resuspending in 1 x PCR
Buffer II (Applied Biosystems), 2 mM MgCla, 1 mM dATP and 0.05 U AmpliTaq per p l and mcubatlng at about 72 ~C for about 15 min. The PCR product is ligated into pBI~-RSV (Stratagene) that has been digested with S~~aI and T-tailed using dTTP and AmpliTaq. The insert is sequenced on both strands by automated fluorescent DNA sequencing (Vanderbilt Cancer Center).
The cycling conditions for the 3' RACE reaction are about 95 ~C, about 1 m in for about one cycle; about 94 ~C, about 5 s, about 72 ~C, about 3 min for about five cycles; about 94 ~C, about 5 s, about 70 ~C, about 10 s, about 72 ~C, about 3 min for about five cycles; about 94 ~C, about 5 s, about 68 ~C, about 10 s, about 72 ~C, about 3 min for about 32 cycles; and about 72 ~C, about 10 min for about one cycle. The product of this reaction is A-tailed, subcloned and sequenced as for the 5' R ACE product.
B. GENERATION OF THE OPEN REALIING FRAME FOR OCTOPAMINE
RECEPTOR (Pa onl) Oligonucleotides used to amplify the open reading frame are a sense oligonucleotide 5' CAGGAATTCATGAGGGACGGGGTTATGAACGCTAG 3' (SEQ ID NO: 8), and an antisense oligonucleotide 5' GCTTCTAGATCACCTGGAGTCCGATCCATCGTTG 3' (SCQ ID NO: 9) Sequences corresponding to the open reading frame are underlined, The sense oligonucleotide contains an EeoR1 restriction site and the antisense oligonucleotide an.~'baI
restriction site, These oligonucleotides are used in a polymerase chain reaction that included the 5'RACE cDNA as template and VENT polymerase (New England Biolabs, Beverly, MA), The product is subcloned into the plasmid pAcS.l/VS-His (Invitrogen Life Technologies, Carlsbad, CA) at the EcoRI and Xl~czl restriction sites and sequenced. This plasmid is designated pAc-Pa oar, For mammalian cell expression, a Kozalc sequence is inserted using a sense oligonucleotide 5'ACAGAATTCGCCACCATGAGGGACGGGGTTATGAACGCTAG 3' (SEQ
ID NO: 10) and an internal antisense oligonucleotide that contains an Xlrol site 5' TTGAGGGCGCTCGAGGACGTC 3' (SEQ ID NO: 11), The sense oligonucleotide contains an EcoRl site, These oligonucleotides are used in a polymerase chain reaction that includes pAc-Pa oar as template and VENT polymerase_ The product is inserted at EcoRI and ~Yhol sites into pAc-Pa oal, in which the corresponding EcoRl and .~hol fragment have been removed, The product is sequenced, The entire open reading frame is then transferred into pCDNA3 (Invitrogen Life Technologies, Carlsbad, CA) at Ec~RI and Apal restriction sites, and this plasmid is designated pCDNA3-Pa oa,.
C. AMPLIFICATION AND SUBCLONING OF OAMB, AN OCTOPAMINE
RECEPTOR FROM THE FRUIT FLY, .UROSOPIIILA MELANOGASTER
The Drosophtla nxela~rogastef° head cDNA phage library GH is obtained through the Berkeley Drosophila Genome Project (www.fruittl .~), Phage DNA is purified from this library using a liquid culture lysate as described in Lech, Current Protocols in Molecular Bioloay, John Wiley & Sons, Inc., pp. 1 (2001), Oligonucleotides designed to amplify the open reading frame of D~°osoplzila Jraelayzogastef~ OAMB C011S15t of the sense oligonucleotide 5' CAGGAATTGGCCACCATGAATGAAACAGAGTGCGAGGATCTC 3' (SEQ ID NO: 12) and the antisense oligonucleotide 5' AATGCGGCCGCTCAGCTGAAGTCCACGCCCTCG 3' (SEQ
ID NO: 13), Sequences corresponding to the open,, reading frame are underlined. A I~ozalc sequence is included in the sense oligonucleotide In addition, the 5' oligonucleotide includes an EcoRl restriction site and the 3' oligonucleotide a Notl site, For amplification by the polymerase chain reaction, about 200 ng of the GH
library DNA
is used as template with about 0.5 EiM of each oligonucleotide and VENT DNA
polymerase (New G
England Biolabs). Cycling conditions are about 95 C, about 5 min for about one cycle; about 95 ~C, about 30 s and about 70 ~C, about 1.5 min for about 40 cycles; and about 70 ~C, about 10 min for about one cycle. The product is digested with EcoRI and Notl, ligated into pCDNA3 and sequenced on both strands by automated fluorescent DNA sequencing (Vanderbilt Cancer Center).
D. ISOLATION OF cDNA ENCODING OCTOPAMINE RECEPTOR (Pa oal) A polymerase chain reaction with degenerate oligonucleotides corresponding to regions of TM VI and TM VII of previously identified octopamine receptors is used to isolate an approximately 101 nucleotide fi~agment of cDNA from the head of Per~ipdaneta aYne~~ieavra. This eDNA fragment is used to design gene specific oligonucleotides to amplify the frill-length eDNA
of the corresponding gene by RACE, This method generates overlapping S' and 3' segments that include the original cDNA fi~agment from TM VI to TM VII indicating these segments originate from the same cDNA, The eDNA includes an approximately 1887 nucleotide open reading frame and 5' and 3' untranslated regions (Genbanl: accession number is AY333178), The predicted initiation codon is preceded by an in-frame stop codon, indicating that the 5' end of the open reading frame is included in the cDNA and that the encoded protein will be full length. This cDNA and encoded protein are designated Pa oar.
The open reading frame encodes a protein of approximately 628 amino acids with a predicted molecular mass of about 68,642 Da. Hydropathy analysis by the method described in I~yte et al., J. Mol. Biol. 157, 105-132 (1982), with a window of about nine amino acids indicates about seven potential transmembrane spanning damains, In addition, a protein BLAST search fords similarity of Pa oat to the rhodopsin family of 7 transmembrane G
protein-coupled receptors contained within the conserved domain database, The BLAST analysis also indicates Pa oar, is most s imilar to other biogenic amine receptors, As melitioned above, all members of GPCT~s share the common motif of seven transmembrane (TM) domains. Of these seven domains, TM 3, 5 and 6 comprise the binding sites. Compared to proteins with defined functions, Pa oar is most closely related to OAMB, an octopamine receptor from the fruit fly 17~~osophila ~rzelafzogaster~ and to Lym- oar, an octopamine receptor from the pond snail Lyn~f~aea stagrralis), Sequence similarity is also detected with vertebrate alA adrenergic receptors and invertebrate tyramine receptors, Protein alignment indicates Pa oal is about 51% identical to OAMB, 37% identical to Lym oar, and about 27%
identical to both the insect tyramine receptors Tyr-Loc from Locusta migi~atoi~ia and Tyr-Dro from D~osoplzila n~elarlogaste~°, Sequence conservation between Pa oar, OAMB and Lym oai, is greatest within the TM domains, as shown in Figure 2. The regions of lowest similarity among these three proteins are in the amino terminus extending into TM l, extracellular loop 2 (between TM IV and V), intracellular loop 3 (between TM V and VI) and the carboxyl termini following TM VII.
E. CELL CULTITRE AND TRANSFECTION OF CELLS
Cell culture reagents may be obtained from Sigma-Aldrich (St Louis, MO), or as otherwise indicated, African green monkey kidney COS-7 cells and human embryonic kidney (HEK)-293 cells are obtained from American 'Type Culture Collection (Manassas, VA). COS-7 cells are grown in Dulbecco's modified Eagle's medium (about 4.5 g glucose/I) and about 10%
fetal bovine serum, HETC-293 cells are grown in Dulbecco's modified Eagle's medium (about 1 g glucose/1), about 5% fetal bovine serum and about 5% newborn calf serum, Both types of media are supplemented with about 100 U penicillin G/ml, about 100 pg streptomycin/ml and about 0.25 pg amphotericin B/ml) except during Lipofectamine 2000 transfections.

Lipofectamine 2000 and Opti-MEM I media may be obtained from Invitrogen Life Technologies (Carlsbad, CA), COS-7 cells are transiently transfected using Lipofectamine 2000.
Cells are plated at about 1.5 x 10G cells per dish (about 55 cm2) in about 10 llll gl'OWth 11'ledlLllll without antibiotics the day before transfection, For each dish, about 30 p1 Lipofectamine 2000 in about 1 ml Opti-MEM I medium is mixed with about 12 pg plaslnid DNA in about 1 ml Opti-MEM I medium and added to the cells after an approximately 20 min incubation at 1'00111 temperature. The cells are harvested for membrane preparation 24 h following transfection.
For stable transfections of HEK-293 cells, about 1 x 10~' cells in about 2.5 ml growth media without antibiotics are plated into dishes (about 10 cm') the day before transfection, For transfection, about 10 p1 Lipofectamine 2000 is added to about 250 p1 Opti-MEM
I medium. This is mixed with about 4 pg plasmid DNA in about 250 p1 OptiMEM I medium. After an approximately 20 min incubation at room temperature, the approximately 500 EII
of solution is added to cells in a single dish, Cells are split about 24 h after transfection into growth media containing about 0.8 mg 6418 sulfate/ml (Mediatech II1C., Heradon, VA), Clonal lines are selected and assayed for receptor expression with whole cell binding by incubating about 500,000 cells in about 1 ml phosphate buffered saline (PBS; 137 mM NaCI, 2.7 mM KC 1, 10 mM
Na2HP0~, 1.4 mM ICI-IZP04 (pH 7.4)) with about 2 nM 3H-yohilnbine for about 30 171111 at about 27 ~C, Cells are pelleted by centrifugation, washed with PBS, and then transferred to scintillation vials. Nonspecific binding is determined by including about 50 yM phentolamine in the binding reaction.
F. EFFICACY OF CELLS LINES TRANSFECTED WITH OCTOPAMINE
RECEPTORS FOR SCREENING COMPOUNDS AND COMPOSITIONS FOR
OCTOPAMINE RECEPTOR INTERACTION
1 (i All steps are performed at about 4 C or on ice, Cells are harvested in growth media by scraping from the dishes and then rinsing dishes with PBS, The cells are centrifuged at about I OOOg for abOLlt 3 111111, washed with PBS and centrifilged again, The cells are suspended in ice cold hypotonic buffer (10 mM Tris-CI (pH 7.4)), incubated on ice for about 10 min, and lysed using a glass Bounce homogenizer and tight glass pestle (I~ontes Glass Co., Vineland, NJ) with about 10 strokes, Nuclei are pelleted by centrifugation at about 600g for about 5 111111, The supernatant is decanted and centrifuged at about 30,OOOg for about 30 min to pellet a crude membrane fraction, The pellet is suspended, in binding buffer (50 mM Tris-C1, 5 mM MgCl2 (pH
7.4)), Protein concentration is determined by the Bradford assay (Bio-Rad Laboratories, Hercules, I 0 CA), Membranes are frozen on dry ice and stored at about -75 ~C in aliquots.
Antagonists and biogenic amines are obtained from Sigma-Aldrich (St, Louis, MO).
Octopamine is the mixed isomeric form DL-octopamine, 3H-yohitnbine is obtained from Perkin Elmer Life Sciences (Boston, MA), Radioligand binding is performed with about 7.5-IS yg membrane protein inabout250 y1 binding buffer for about 30 min at about 27 ~C
while shaking at about 100 rpm, Reactions are terminated by addition of abaut 3 ml ice cold binding buffer and filtered over GF/C filters (Whatman International, Maidstone, England) that have been soaked for about 30 min in about 0.3% polyethylenimine (Sigma-Aldrich), Filters are rinsed again with about 3 m1 binding buffer, For the determination of ICS; and B",a~, a range of 3H-yohilnbine is used from about 0.5 to 50 nM, and about 50 ~,M phentolamine is used as a competitor to determine nonspecific binding, To determine K;, of different ligands, about 2 nM 3H-yohimbine is used with a concentration range of competitor that gives from 0% to 100% competition, Binding data is analyzed by nonlinear regression using the software GraphPad Prism (San Diego, CA), For pharmacological binding experiments, Pa oa;, is expressed in COS-7 cells by transient transfection. Membrane fractions are analyzed to determine total, nonspecific and specific binding of 3H-yohimbine, as shown in Figure 3A. The Kd and B",~X for specific binding are determined to be about 28.4 nM and about 11.8 pmol/mg protein, respectively.
Membrane fractions from COS-7 cells transiently transfected with empty pCDNA3 do not demonstt ate specific binding. The high affinity binding of 3H-yohimbine by Pa oa; indicate that this is a suitable ligand to be used for competition binding experiments.
The octopamine receptor OAM:B from DJ°osophila n~elaroogastef°
is ampliFed by the polymerase chain reaction. Saturation binding analysis with 3H-yohimbine is performed with OAMB expressed in GOS-7 cells, as shown in Figure 3B. The I~~ and B",ax are determined to be about 43.0 nM and about 8.04 plllol/111~, respectively.
Competitive binding with various biogenie amines is utilized to determine the affinities for potential natural ligands of Pa oal. Referring now to Table A, below, DL-Octopamine has the lowest I~; (about 13.3 p,M) for Pa oat followed by tyramine (about 31.0 p.M).
The decreasing order of affinity for the biogenic amines is octopamine > tyramine > dopamine > serotonin. The binding affinities for octopamine and tyramine are determined for this receptor. The K; (mean ~
standard deviation) of octopamine and tyramine for OAMB are about 8.20 ~2.60yM
and about 33.8 ~ 7.93 wM, respectively. These values are similar to those obtained for Pa oa;. The affinity of octopamine is about 2.3-fold higher than tyramine for Pa oa;, and for OAMB, the affi pity of octopamine is about 4.1-fold higher than tyramine, indicating that octopamine is the likely endogenous ligand for Pa oa;.
Liga.nd I~; (yM) Bioge~ic As~tihe Octopamine 13.3 ~ 2.4 Tyramine 31.0 ~ 1.9 Dopamine 56.6 ~ 8.0 Serotonin 77.4 ~ 11.6 AYl tagOl2lSl Chlorpromazine ' 0.012 ~ 0.003 Phentolamine 0.023 ~ 0.009 Mianserin 0.048 ~ 0.013 Metoclopramine 4.76 ~ 1.32 Table A
In addition to using the affinity of octopamine receptors for specific antagonists as a method for classifying these receptors, antagonists may be used to analyze the effects of octopamine on adenylate cyclase activity in the brain, ventral nerve cord and hen iocytes of Penipla~reta arne~ieana. A pharmacological profile is developed for Pa oal using these antagonists. With reference to Table A, in order of decreasing affinity, the profile of the antagonists is chlorpromazine > phentolamine > mianserin > metoclopramide.

STRUCTURAL FEATURES OF CLONED AMERICAN COCKROACH OCTOPAMINE
RECEPTOR (Pa oa,) The Pa oar cDNA of 2268 by which includes an 1887 nucleotide open reading frame and 5' and 3' untranslated regions is set forth in Figures 1A, 1B and SEQ ID NO:
1. With reference to Figure 1B, the predicted initiation codon (M) is preceded by an in-frame stop codon (SC). This indicates that the 5' end of the open reading frame is included in the cDNA
and that the encoded protein would be full length.
With reference to Figure 4, hydropathy analysis by the method of Kyte and Doolittle with a window of 9 amino acids indicates that this sequence shares the common motif of 7 potential transmembrane scanning domains. See I~yte and Doolittle, 1982, J. Mol. Biol.
157, I05-132. A
phylogenic comparison of invertebrate biogenic amine receptor sequences reveals that both OAMB and Pa oa, sequences share ~ 45% similarity, which is illustrated in Figure 5. Pa oar clusters with octopamine and tyramine receptors from different insect species.
Similarity between 1p these receptors is analyzed using BLAST search aiid calculated based on protein alignment using DNASTAR software program. Pa oal is used as a reference for comparisons with other receptors.
With reference to Figure 2, protein alignment indicates sequence conservation between Pa oal and OAMB is greatest within the transmembrane domains (TMs). The regions of lowest similarity among these two proteins are in the amino terminus extending into TM1, extracellular loop2 between TM4 and TMS, intracellular loop between T-'MS and TM6 and the carboxy termini following TM7.

EFFECTS OF TREATMENT WITH OCTOPAMINE ON CELLS EXPRESSING THE
OCTOPAMINE RECEPTOR (Pa oal~
A. EFFECT OF TREATMENT ON [CAMP]
Twenty-four hours before cell treatment, about 300, 000 I-IEK-293 cells are plated in about 1 ml media with about 0.8 mg G418/ml into multi-well dishes (e.g., 12-well, 4.5 cm'). For cell treatment, the media is aspirated and about 1 ml PBS with about 300 pM IBMX
and the test reagent is added. Cells are incubated at about 37 ~C for about 20 min, and the PBS is then aspirated. Cells are incubated with about 70% ethanol for about 1 h at about -20 ~C. The cellular debris is centrifiiged and then the supernatant is removed and lyophilized to dryness. The amount of CAMP in the extract is determined by using a cAMP binding protein fro111 the ~H-cAMP
Biotralc assay system (Amersham Biosciences) as per the manufacturer's instructions. To test the effects of calcium chelation on cAMP levels, the cells are incubated with about 20 y 1 V 1 BAPTA/AM (Calbiochem Biochemicals, La Jolla, CA) for about 10 min before the addition of the test reagents.

Octopamine has been demonstrated to increase levels of the second messenger cAMP in brain, thoracic ganglion and hemocytes from Peoiplaa2eta arne~~ica~a. To deterlllllle WhlCh SeCOlld messenger signaling pathways octopamine could affect through the Pa oal receptor, HEIR-293 cells are stably transfected with pCDNA3-Pa oat or pCDNA3 without an insert as a control In the control HEIR-293 cells, neither DL-octopamine nor tyramine at concentrations up to about 100 pM has significant effects on CAMP levels, A clone transfected with pCDNA3-Pa oal having a high specific binding t0 3H-yohl111b111e is selected for second messenger analysis, BOth octopamme alld tyramine are able t0 increase the levels of cAMP in these cells in a dose dependent manner, as shown in Figure 6, The >JCSOS for the octopamine and tyramine mediated increases in cAMP are about 1.62 and 97.7pM, respectively (p < 0.05). Octopamine is more potent than tyramine in the cAMP
response as a statistically significant increase in CAMP over the basal level (about 0.48 pmol CAMP) is first detected with about 10 11M octopamine (about 1.2 pmol CAMP) (p < 0.05). The cAMP
concentration with about 10 nM tyramine is about 0.50 pmol CAMP, and therefore not statistically significant from the basal level (p > 0.05), A concentration of about 1 EIM
tyramine results in an increase in CAMP to about 1.2 pmol. In addition, about 100 IIM octopamine leads to an approximately 911-fold increase in cAMP compared to an approximately 215-fold increase for about 100 p,M tyramine. Since these assays are performed in the presence of the phosphodiesterase inhibitor IBMX, the increases in CAMP is determined to be through activation of adenylate cyclase. As such, it appears that the Pa oal receptor is an octopamine receptor, the Pa oal receptor may be targeted to effect a disruptive change in intracellular levels of cAMP, controlled targeting of the receptor allows for insect control, and the cell lines stably expressing the Pa oal receptor may be used to screen compounds and compositions for insect control activity.

B. EFFECT OF TREATMENT ON cAlVn' AND (Ca2+]
To determine cAMP levels in cells, about 24-hovers before cell treatment, 300,000 HEK-293 cells are plated in 1 mL media with 0.8 mg G418/n~L lllt0 111L11t1-dlSheS
(4.5 cm2). For cell treatment, the media is aspirated and 1 mL PBS with 3~0 p.M IBMX and the test reagent is added.
Cells are incubated at 37°C for 20 min, and the PBS is then aspirated.
Cells are incubated with .
70°e° ethanol for 1 hour at-20°C. The cellular debris is centrifuged and then the supernatant is removed and lyophilized to dryness. The amount of c~MP in the extract is determined by using a cAMP binding protein from the 3H-cAMP Biotrak assay system (Amersham Biosciences, Piscataway, NJ) as per the manufactzlrer's instructions.
To determine Ca2+ levels in the cells, HEK-293 cells are washed once with Hank's balanced salt solution (137 mM NaCI, 5.4 mM ICCI, 0.3 mM NaZHP04, 0.4 mM
ItH~P04, 4.2 mM NaHC03, 1 mM CaCl2, 1 mM MgSO4, and 5.6 mM glucose (pH 7.4)) (HBSS). Cells are collected by scraping and are suspended at about 750,000 cells/ml in HBSS with about 5 p.M
Fura-2 AM (Sigma-Aldrich), Cells are incubated at abo ut 37 ~C for about 1 h in the dark, centrifuged, suspended in HBSS at about 750,000 cellsJml and used for calcium measurements A
spectrofluol°emeter with Felix software from Photon Te chnology International (Lawrencevi Ile, NJ) is used for the fluorescence measurements and data- collection, Octopamine has been demonstrated to modulate; intracellular calcium levels in cultured hemocytes of Malacosonza disstria. Also, in hemocytes from Peg~ipla~eta annericafna, octopamine lead to an increase in inositol triphosphate which likely will lead to increases in calcium in these cells as well, The ability of both octopamine and tyran W na to modulate calcium levels in the 1-IEK-293 clone expressing Pa oat is determined, Neither about 10 yM octopamine nor about 10 pM
tyramine modulates intracellular calcium levels in collt~'ol HEIR-293 cells transfected with pCDNA3 lacking an insert, However, when about 100 r1M octopamine is added to the Pa oa, 2?

expressing HEIR-293 cells, a rapid increase in intracellular calcium is detected, as shown in Figure 7. In these same cells, about 100 nM tyramine does not modulate intracellular calcium levels, as shown in Figure 7, Testing of these amines at additional concentrations indicates that the lowest concentration of octopamine that increases intracellular calcium levels is about 10 nM.
Tyramine is found to increase intracellular calcium when a concentration of about 1 p.M or higher is tested, These increases in intracellular calcium by about 10 nM octopamine and about 1 pM
tyramine are to a similar level, both of which is lower than the increase in calcium mediated by about 100 nIVI
octopamine, This result is similar to that obtained with the cAMP assay in that an approxim ately 100-fold increase in tyramine concentration compared to about 10 nM octopamine is required to give a similar level of response, As such, it appears that the Pa oar receptor.is an octopamine receptor, the Pa oar rec eptor may be targeted to effect a disruptive change in intracellular levels of Cap+, controlled targeting of the receptor allows for insect control, and the cell lines stably expressing the Pa oar receptoa- may be used to screen compounds and compositions for insect control activity.
Octopamine is found to increase both cAMP and calcium in HEK-293 cells expressing Pa oar and the calcium increase is detected immediately upon octopamine addition.
As such, tL-~e possibility that calcimil is leading to a secondary increase in cAMP levels in the cells expressing Pa oar is tested. The intracellular calcium chelator BAPTA/AM is used, BAPTA/AM at abut 20 pM is found to inhibit the increase in free intracellular calcium when about 1 yM octopami 3~e is added to the Pa oar-expressing cells, Octopamine-mediated changes in cAMP
levels are cotmpared in the absence and presence of about 20 p.M BAPTA/AM, cAMP levels following treatment with either about 100 nM or about 1 ~,M octopamine, as well as basal cAMP levels, are not four d to be statistically different, whether in the absence or presence of about 20 ~M
BAPTA/AM, as shown in Figure 8. This indicates that the increase in CAMP by octopamine results from direct coupling of Pa oar to a G protein that leads to activation of adenylate cyclase, malting the expression of Pa oa; in HEK-293 cells a good model for adenylate cyclase-modulated insect control through this receptor and the cell lines stably expressing the Pa oat receptor useful for screening compounds and compositions for insect control activity.

RECEPTOR BINDING AND CHANGES IN cAMP AND INTRACELLTJLAR CAZ+ IN
RESPONSE TO OCTOPAMINE TREATMENT
For radioligand binding studies, the binding of 3H-yohimbine to membranes isolated from COS-7 cells expressing Pa oa; and octopamine receptor'(OAMB) from 17r°osoplaila ~raela~rogaste~°
Are performed. See Bischof and Enan, 2004, Insect Biochem. Mol. Biol. 34, pp.
511-521, which is incorporated herein by this reference. The data shown in Table B
demonstrates that the affinity of Pa oa; to the radioligand is abaut 1.5 fold higher than OAMB. Radioligand binding using 3H-yohimbine is performed on membranes expressing either either Pa oar or OAMB.
For the determination of ICd and B",~, a range of 3H-yohimvine is used from 0.5 to 50 n M, and 50 yM
phentolamine is used as a competitor to determine nonspecific binding. To determine IC; of octopamine, 4 nM 3H-yohimbine is used with a concentration range of octopam ine that gives from 0 to 100% competition.
OAR Species I~d B",a, Ki (nM) (pmole receptor/mg (yM) protein) OAR species 28.4 11.80 13.30 OAMB 43.0 8.04 8.20 Table B
With reference to Figure 9, OA (10 pM) increases the level of cAMP in HEIR-293 cells permanently expressing either OAMB or Pa oar. With reference to Figures 10A
and l OB, OA (10 l.~M) increases the level [Ca'+]; in HEK-293 cells permanently expressing either OAMB or Pa oa;, where HEK-293 cells expressing either receptor are incubated for 30s before the addition of 10 1.~M octopamine (OA). The arrow in the figures indicates addition of the amine. The fluorescence ratio determined from excitation with 340 and 380 nm is plotted to indicate changes in [Ca''+];
levels. These increases are mediated through the OAR as judged by the insignificant changes in cAMP level and [Ca2~]; in cells transfected with an empty vector then treated with 10 yM OA
(data not shown).

EXPRESSING THE OCTOPAMINE RECEPTOR
In this example, membranes isolated from COS-7 cells expressing the receptor are used for receptor binding studies and HEK-293 cells are.used for CAMP and [Ca'+];
studies. Plant 15 essential oils, including: ~-cymene [methyl(I-methylethyl)benzene], eugenol [2-methoxy-4-(2-propenyl)phenol], traps-anethole [I-methoxy-4-(1-propenyl)benzene], cinnamic alcohol [3-phenyl-2-propen-1-of], a,-terpineol [p-menth-I-en-8-of], methyl salicylate [2-hydroxybenzoic acid methyl ester], 2-phenylethyl propionate, and geraniol [3,7-dimethyl-2,6-octadien-1-of], are obtained from Gity Chemical (West Haven, CT) and tested for insect control activity. The 20 chemical structures of these compounds are set forth in Figure 11.
A. RECEPTOR BINDING ACTIVITY
The binding activity of 3H-yohimbine to membranes expressing Pa oa; or OAMB is performed in the presence and absence of three structurally related plant essential oil 25 monoterpenoids, which are selected based on their insecticidal activity, the absence or presence and location of the hydroxyl group and a spacing group within the molecule.
Membrane protein (10 yg) expressing Pa oa; is incubated with 4 nM 3H-yohimbine in the presence and absence of 50 25.

p,M of the test chemical. The specific activity is calculated as the difference between counts in the presence and absence of test chemical. Specific binding is calculated by determining nonspecific binding with 50 yM tested plant essential oils and subtracting nonspecific binding fr 0111 total binding.
With reference to Figure 12, depicting specific binding of 3H-yohimbine to Pa oai and OAMB, while eugenol and cinnamic alcohol decrease the binding of 3H-yohimbine to membranes expressing either Pa oar or OAMB as compared to the corresponding control, trans-anethole decreases the 3H-yohimbine binding activity to only Pa oal. It is also found that eugenol and trans-anethole are more potent inhibitors against Pa oar than OAMB, while cinnamic alcohol is more potent against OAMB than Pa oal. The data suggested insect species differences in receptor binding in response to monoterpenoids.
B. EFFECTS OF TREATMENT ON [CAMP]
Figure 13 depicts the effect of certain plant essential oils on cAMP levels in cells expressing either Pa oai or OAMB. HEIR-293 cells stably expressing either receptor are treated with 300 ECM IBMX and the effect oftested plant essential oils (50 p.M) on basal CAMP
levels is measured.
Eugenol (50 ~~M) significantly decreases the cAMP level (24%) in cells expressing Pa oar but slightly decreased cAMP level in cells expressing OAMB. A 22% increase in cAMP level in cells expressing OAMB is fOUlld in response to treatment with (SO yM) traps-anethole. Cinnamic alcohol (50 p.M) induces slight increase in CAMP level in both cell models.
C. EFFECT OF TREATMENT ON INTRACELLULAR CALCIUM
MOBILIZATION
To address whether changes in [Ca2+]; in octopamine receptor-expressing cells in response to 25 yM of tested plant essential oils is mediated specifically through the receptor, cells 2 (~

transfected with an empty plasmid (pCDNA3) are treated with either test chemicals or solvent only and changes in [Ca2+]; are monitored. In cells transfected with an empty plaslnid, none of the test chemicals induce remarkable changes in [Cap+]; levels as compared with cells treated with the solvent (data not shown).
On the other hand, changes in [Ca2+]; level in cells expressing either OAMB or Pa oa; in response to test chemicals is remarkably high. Figures 14A-14F, depict the effect of cinnamic alcohol (Figures 14A and 14B), eugenol (Figures 14C and 14D), and t-anethole (Figures 14E and 14F) on intracellular calcium [Ca2+]; levels in HEK-293 cells either transfected with Pa oar or OAMB. HEK-293 cells are incubated for 30s before the addition of 25 pM tested agents. The arrow in the figures indicates addition of tested agents. The fluorescence ratio detel'nlllled fl'Olll excitation with 340 mn and 380 nm is plotted to indicate transient increase in [Ca2+]; levels.
Generally, changes in [CaZ~'~]; in cells expressing OAMB is more pronounced than changes in cells expressing Pa oal. Based on increased [Ca~''+]; level in cells expressing OAMI3, cinnamic alcohol is the most potent agent tested in this example, followed by eugenol and traps-anethole.
In cells expressing Pa oal, eugenol is the most potent agent tested in this example, followed by cinnamic alcohol then traps-anethole. The data suggest that elevation pattern of [Caz-F]; levels is chemical-dependent. While application of octopamine induces an immediate but transient peak (~
sec) in [Ca2+]; level, as shown in Figure 9, the peaked [Ca2+]; level is slower in onset and has a longer recovery time (more than 3 min) in response to treatment with tested plant essential oils.
20 In cells expressing OAMB, the increase in [GaZ+]; level in response t0 C11111a1111C alCOl101 is slower than the other two chemicals. In Pa oa;-expressing cells, the increase in [Ca'+]; in response to traps-anethole is slower than eugenol and cinnamic alcohol. Thus, the efficacy of coupling of both cloned octopamine receptors to different second messenger signaling varies with the chemical used.
D. SUMMARY OF THE EFFECTS OF TREATMENT WITH CERTAIN PLANT
ESSENTIAL OILS
2e The present example studies the molecular interaction of plant essential oils with octopamine receptors fi~om different insect species. Based on the characteristic features of octopamine receptors from American cocla~oach and fruit fly, the example characterizes certain molecular basis for insect species differences in response to plant essential oils. AIthOLIgII trans-anethole does not have a significant effect on bmdmg t0 OAMB while eugenol and cinnamic alcohol do (Figvlre 12), only traps-anethole increases cAMP level (Figure 13) and [Ca''~'~]; (Figures 14A-14F) through OAMB. These findings suggest that, in the case of traps-anethole, ionic interaction between the tested agent and the receptor is not critical for the activation of signaling down stream to OAMB.
On the other hand, while both eugenol and cinnamic alcohol decrease the binding activity to Pa oa; (Figure 12), only eugenol decreases cAMP levels through this receptor (Figure 13).
However, these two chemicals increase [Ca'+]; through Pa oa; and OAMB (Figures 14A-14F).
The data demonstrates that activation of Pa oal by traps-anethole and cinnamic alcohol is not primarily coupled to cyclic nucleotide system. It appears that it is coupled to IP3-system, which activates the release of Ca2+ ions from internal stores. Activation of Pa oa;
by eugenol is Found to be coupled to both adenylate cyclase/cAMP and IP3/Ca2+ signaling cascades.
Therefore, the current changes in cellular responses suggest that tested plant essential oils differing by only a single hydroxyl group or methoxy group in their chemical structure are capable of differentially coupling each octopamine receptor to different second messenger systems. The data also suggest that, activation of single GPCR such as Pa oal or OAMB, may potentially couple to multiple second messenger systems. Thus, a single receptor may have a different pharmacological profile depending on which second messenger system is activated. The variability of the transmembrane regions and N-termini of Pa oal and OAMB might determine the selectivity of tested monoterpenoids. In addition, conservation of cel-tain transmembrane motifs and the variability of the intracellular loops might enable Pa oar and OAMB to discriminate among the various G-protein subtypes upon treatment with tested monoterpenoids.
Protein alignment indicate that the regions of lowest similarity among these two proteins ar a in the amino terminus extending into TM1, extracellular loop2 between TM4 and TMS, intracellular loap between TM5 and TM6 and the carboxy termini following TM7 (Figure 2). On the other hand, protein alignment indicates sequence conservation between Pa oai and OAMB is greatest within the transmembrane domains (TMs).

TOXICITY TESTING AGAINST CERTAIN INSECT SPECIES
Toxicity bioassay against the wild type Drosophila nzelan~gaster fly and American cockroach is performed to address insect species 5peciticity in response to certain plant essential oils and to determine whether the cellular changes in Pa oar and OAMB cell models in response to treatment with tested essential oils correlate with their insecticidal activity.
Dnosophila n-aelaaogaste~ wild type strain is purchased from Carolina Bialogical Supply Company (Burlington, NC). Flies carrying the inactive (iav) mutation that exhibit low locomotor activity and poor mating success, both of which are associated with a deficiency in octopamine synthesis are obtained from Bloomington Drosopl7ila Stocl: Center (flybase ID
FBaI 0005570, stoclc# BL-6029 iav).
Plant essential oils, including: p-cymene [methyl(1-methylethyl)benzene], eugenol [2-methoxy-4-(2-propenyl)phenol], traps-anethole ~l-methoxy-4-(1-propenyl)benzene], cinnamic alcohol [3-phenyl-2-proper-1-of], a,-terpineol [p-month-1- en-8-of], methyl salicylate [2-hydroxybenzoic acid methyl ester], 2-phenylethyl propionate, and geraniol [3,7-dimethyl-2,6-octadien-1-of], are obtained from City Chemical (West Haven, CT) and tested for insect control activity. The chemical structures of these compounds are set forth in Figure 11.
2~~

Acetonic solutions of plant essential oils are prepared and different concentrations of each, that give from 10% - 100% mortality, are applied by topical application.
Controls are treated with the same volume (0.5 yl/insect) of acetone. Replicates, with 5 insects per replicate, are used for each concentration. The mortality is calculated 24 hours after treatment. Data are subjected to probit analysis to determine LDSO value for each compound. See Finney, 1971, Probit Analysis, 3'd Ed., Cambridge University Press, London, pg. 333.
To determine whether the octopamine/octopamine receptor (OA/OAR) system is involved in the toxicity of tested plant essential oils, octopamine synthesis mutant (iav) DT~o.s~oplaila mela~ogaste~~ strain is topically treated with a dose eduivalent to the determined LDSO for wild type strain. For this study, the LDSO values of eight monoterpenoid plant essential oils (p-cymene, eugenol, traps-anethole, cinnamic alcohol, a-terpineol, methyl salicylate, phenylethyl propionate, and geraniol) are determined against wild type as described above and being used to treat the octopamine mutant (iav) fruit fly. Controls are treated with the same volume (0.5 E~l/fly) of acetone. The mol-tality is calculated 24 hour after treatment. Multiple replicates and 5 flies per replicate are used for the bioassay of each chemical. Data are subjected to probit analysis to determine LDso value for each chemical. See Finney, 1971.
To determine insect species differences in response to plant essential oil 1110170terpel'lOldS, the toxicity of certain monoterpenoids is determined against fruit fly and American cockroach. Based on the calculated LDSO values, shown 111 Table C, cinnamic alcohol is the most toxic chemical tested in the example (LDSO = 1.65 ~.g/fly) against wild type fruit fly strain, followed by eugenol (LDso = 1.90 p.g/fly), and traps-anethole (LDSO = 6.00 l.lg/fly). Cugenol is about 2-Fold and about 27-fold more toxic against American cockroach than cinnamic alcohol and traps-anethole, respectively.
Plant essential oil ~ D. >'r2ela~ogastey~ ~ P. Anaericarza Cirlnamic alcohol 1.G5 98 Eugenol 1.90 47 Traps-anethole 6.00 1300 Table C
To determine whether the OA/OAR system mediates the toxicity of certain plant essential oil monoterpenoids, fruit flle5 CaTlylllg the dCIV IllLItat10115, which are highly susceptible to the octopalnine analoguep-cresol, are used in parallel with wild type fruit fly strain in the taxicity bioassay test. The toxicity Of CII111a1111C alCOh0l, eugenol, traps-anethole and 2-phenyethyl propionate is remarlcably increased when they are topically applied to the ierv strain, as shown in Table D.
Wild/type %Mortality at LDSO of wild/type Chemical name LDso valuesDrosophila melczsaogaster strain (l.~g/ily) Wild/type ierv cinnamic alcohol 1.G5 30.0% 80.0!

eugenol 1.90 53.3% 80.0%

traps-anethole 6.00 40.0% 100.0%

methyl salicylate 7.50 40.0% 4G.G%

geraniol 10.50 G0.0% G0.0%

a-terpineol 13.00 46.G% G0.0%

2-phenylethyl propionate14.50 53.3% 80.0%

p-cymene 25.00 40.0% 40.0%

Table D

However, mutation of the octopamine synthesis does not affect the toxicity ofp-cymene, methyl salicylate, and geraniol. Therefore, the current data suggests a correlation between agents 111d11Clllg Celhllar Changes 111 CIOllal cells expressing octopamine receptors and their insecticidal activity. The data also suggests that the insecticidal activity of cinnamic alcohol, eugenol, trans-anethole and 2-phenyethyl propionate is mediated through the octopamine/octopamine receptor system. From these data it can be concluded that the increase in the insecticidal activity of these chemicals results from the deficiency of octopamine synthesis in iav mutants because low octopamine levels may be unable to compete against the toxic effect of these chemicals.
As mentioned above, the toxicity data demonstrates significant differences between the toxicity of the tested chemicals against each insect (Table C). The toxicity data also demonstrates differences between the two insects in response to each chemical. The toxicity data against wild type and octopamine mutant (iav) fruit fly suggests that the toxicity of cinnamic alcohol, eugenol and traps-anethol is mediated through octopamine/octopamine receptors system.
Among certain other plant essential oils tested against both strains of fruit fly only the toxicity of 2-phenylethyl propionate is mediated through octopamine receptors. Collectively the data suggest a correlation between cellular changes and toxicity of certain plant essential oils.
In the present example, chemical-structure relationships of plant essential oil monoterpenoids against wild type fruit fly suggest certain structural features required for chemical-receptor interaction. Among these features are the presence and location of a hydroxyl group, and a spacing group such as lnethoxy group. The rank order of toxicity demonstrates that cyclic alcohols and phenolic compounds are more toxic than other monoterpenoids such as acyclic alcohols and esters. The efficacy of each compound is found to be determined by the presence and location of the spacing group on the benzene ring. For example, although the phenolic derivative, eugenol, and propenyl benzene, traps-anethol, contain the same spacing group (-32.

OCH3) on position 2 and l, respectively, eugenol is 3-fold more toxic against wild type flies than traps-anethole (Figure 11 and Table D).
In summary, the similarities and differences between both Pa oar and OAMB
sequences are determining features in the toxicity differences of certain plant essential oil monoterpenoids.
Additionally, it appears that the octopamine receptor mediates the insecticidal properties of cinnamic alcohol, eugenol, traps-anethole and 2-phenylethyl propionate and, in part, the toxicity of a-terpineol against Droso~hila fzzelanogaste~° fly. Furthermore, it appears that the presence of an electronegative group SLICK as hydroxyl group, and different spacing groups, may be required for the insecticidal activity of plant essential oils through octopamine receptor.
'~ ~' a' It will be apparent to those skilled in the art that various modifications and variations can be made in the present ITlVelltloll Wlthollt departing from the scope or spirit of the invention. It is intended that the Specification and Example be considered as exemplary only, and not intended to limit the scope and spirit of tile invention. The references and publications cited herein are incorporated herein by this reference.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the Specification, Examples, and Claims are to be understood as being modified in all instances by the term "about."
Accordingly, unless indicated to the contrary, the numerical parameters set forth in the Specification, Example, and Claims are approximations that may vary depending upon the desired properties sought to be determined by the present invention.

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Claims (31)

1. ~A strain of cells comprising a DNA vector having a nucleic acid sequence consisting essentially of SEQ ID NO: 1.

2. ~The strain of cells of claim 1, wherein the cells are COS-7 cells or HEK-293 cells.

3. ~A strain of cells expressing a protein having the amino acid sequence of SEQ ID
NO: 2, or a fragment or derivative thereof, wherein the fragment or derivative thereof binds octopamine.

4. ~A strain of cells expressing a protein having the amino acid sequence of SEQ ID
NO: 2.

5. ~The strain of cells of claim 4, wherein the cells are COS-7 cells or HEK-293 cells.

6. ~A strain of COS-7 cells or HEK-293 cells expressing a protein having the amino acid sequence of SEQ ID NO: 3.

7. ~An isolated nucleic acid molecule sequence encoding a protein that binds a biogenic amine, resulting in changes in intracellular concentrations of cAMP, Ca24, or both, having a nucleic acid sequence consisting essentially of SEQ ID NO: 1.

8. ~A molecule of claim 7, wherein the protein has an amino acid sequence consisting essentially of SEQ ID NO: 2.

9. ~An isolated nucleic acid molecule sequence having at least about 30%
similarity to SEQ ID NO: 1, wherein the molecule encodes a protein that binds a biogenic amine, resulting in changes in intracellular concentrations of cAMP, Ca2+, or both.

10. ~The isolated nucleic acid molecule of claim 9, wherein the molecule encodes an octopamine receptor.

11. ~The isolated nucleic acid molecule of claim 10, wherein the molecule encodes a protein having an amino acid sequence consisting essentially of SEQ ID NO: 2, or a fragment or derivative thereof.

12. ~An isolated nucleic acid molecule sequence of SEQ ID NO: 1, or a fragment or derivative thereof, wherein the molecule encodes an octopamine receptor.

13. ~The isolated nucleic acid molecule of claim 12, wherein the molecule encodes a protein having the amino acid sequence set forth in SEQ ID NO: 2, or a fragment or derivative thereof.

14. ~A method of screening compounds and/or compositions for insect control activity, comprising:
providing cells expressing a protein having the amino acid sequence of SEQ ID
NO: 2; or a fragment or derivative therof wherein the fragment or derivative binds a biogenic amine;
adding said compound and/or compositions to the cells; and measuring the effects of the compounds and/or compositions.

15. ~The method of claim 14, wherein the step of measuring the effects of the compounds and/or compositions includes measuring the binding affinity of said compounds and/or compositions to the receptor.

16. ~The method of claim 15, and additionally comprising: selecting compound and/or compositions having an affinity for the receptor.

17. ~The method of claim 14, wherein the step of measuring the effects of the compounds and/or compositions includes: extracting intracellular cAMP and/or Ca2+ from the cells; determining the intracellular cAMP and/or Ca2+ levels; and comparing the intracellular cAMP and/or Ca2+ levels in cells treated with said compounds and/or compositions to the intracellular cAMP and/or Ca2+ levels in untreated cells.

18. ~The method of claim 17, and additionally comprising: selecting compounds and/or compositions, the treatment with which, causes a change in intracellular cAMP
and/or Ca2+ levels.

19. ~A method of screening compounds and/or compositions for species-specific insect control activity, comprising:
providing first cells expressing a first octopamine receptor cloned from a first insect species-of-interest;
providing second cells expressing a second octopamine receptor cloned from a second insect species-of-interest;
adding said compounds and/or compositions to the first and the second cells;
and measuring the effects of the compounds and/or compositions.

20. ~The method of claim 19, wherein the step of measuring the effects of the compounds and/or compositions includes measuring the binding affinity of said compounds and/or compositions to the first and the second receptor.

21. ~The method of claim 20, and additionally comprising: selecting compounds and/or compositions having a desired relative affinity for one of the receptors.

22. ~The method of claim 19, wherein the step of measuring the effects of the compounds and/or compositions includes: extracting intracellular cAMP and/or Ca2+ from the first and the second cells; determining the intracellular cAMP and/or Ca2+
levels; and comparing the change in intracellular cAMP and/or Ca2+ levels in the first cells and the second cells.

23. ~The method of claim 22, and additionally comprising: selecting compounds and/or compositions, the treatment with which, causes a desired relative change in intracellular cAMP
and/or Ca2+ levels in one of the cells.

24. ~The method of claim 19, wherein one of the octopamine receptors has an amino acid sequence of SEQ ID NO: 2, or a fragment or derivative thereof.

25. ~The method of claim 24, wherein one of the octopamine receptors has an amino acid sequence of SEQ ID NO: 3, or a fragment or derivative thereof.

26. ~A method of testing the effects of compounds on cells, said method comprising:

providing cells expressing a protein having the amino acid sequence of SEQ ID
NO: 2; or a fragment or derivative therof, wherein the fragment or derivative binds a biogenic amine;
adding said compounds and/or compositions to the cells; and measuring the effects of the compounds and/or compositions.

27. ~A method of testing the effects of compounds on cells, said method comprising:
providing first cells expressing a first octopamine receptor cloned from a first insect species-of-interest;
providing second cells expressing a second octopamine receptor cloned from a second insect species-of-interest;
adding said compounds and/or compositions to the first and the second cells;
and measuring the effects of the compounds and/or compositions.

28. ~A report produced by a method, comprising:
providing cells expressing a protein having the amino acid sequence of SEQ ID
NO: 2; or a fragment or derivative therof, wherein the fragment or derivative binds a biogenic amine;
adding said compounds and/or compositions to the cells; and~
measuring the effects of the compounds and/or compositions.

29. ~A report produced by a method, comprising:
providing first cells expressing a first octopamine receptor cloned from a first insect species-of-interest;

providing second cells expressing a second octopamine receptor cloned from a second insect species-of-interest;
adding said compounds and/or compositions to the first and the second cells;
and measuring the effects of the compounds and/or compositions.

30. A compound and/or composition for controlling insects identified by a method, comprising:
providing cells expressing a protein having the amino acid sequence of SEQ ID
NO: 2; or a fragment or derivative therof, wherein the fragment or derivative binds a biogenic amine;
adding said compounds and/or compositions to the cells; and measuring the effects of the compounds and/or compositions.

31. A compound and/or composition for controlling insects identified by a method comprising:
providing first cells expressing a first octopamine receptor cloned from a first insect species-of-interest;
providing second cells expressing a second octopamine receptor cloned from a second insect species-of-interest;
adding said compounds and/or compositions to the first and the second cells;
and measuring the effects of the compounds and/or compositions.