# Patent

## Title
Method for pheromone discovery in insects

## Abstract

The present invention is directed generally to a method of identifying an insect pheromone. Initially, a candidate insect pheromone-binding protein is obtained and sequenced. Specific proteins may then be selected by observing the pattern of pheromone-binding protein expression in the insect stage, phase or caste; and/or in the antenna and other sensilla by, for example, in situ hybridization; and/or by comparison with sequence of known pheromone binding proteins. A composition of one or more pheromones may then be contacted with the pheromone-binding protein. Any pheromones bound to the protein may then be eluted and analyzed.

## Background
This present application claims priority to co-pending U.S. Provisional Patent Application Ser. No. 60/376,451 filed on Apr. 30, 2002. The entire text of the above-referenced disclosure is specifically incorporated herein by reference without disclaimer.


BACKGROUND OF THE INVENTION
I. Field of Invention
The present invention relates generally to a method for isolating insect pheromones. More particularly, the present invention relates to a method for purifying pheromone-binding proteins, and then using the purified proteins to capture insect pheromones.
II. Description of Related Art
Insects receive information from external chemical signals by means of receptors located primarily in the antenna. The dendrites of olfactory receptor neurons in the antenna typically are located in sensilla, thin hair-like structures which protrude from the antennal surface (Altner and Prillinger, 1980; Zacharuk, 1985). Receptor neurons appear to be specialized for particular substances, and each olfactory sensilla may contain dendrites from many different receptor neurons. The plasma membranes of the dendrites contain the olfactory receptor proteins (Hildebrand and Shepard, 1997; Krieger and Breer, 1999).
When activated by an odor or pheromone molecule, the receptors couple to G proteins, which in turn alter ion channel conductance in the receptor neuron membrane, mostly via an IP3 pathway (Breer et al., 1990), although a parallel cAMP pathway may also occur (Krieger et al., 1999). The receptor neurons form synapses with interneurons in the glomeruli of the antennal lobe of the brain (Hansson and Anton, 2000). Projection neurons carry the signals from the antennal lobe to the mushroom body, where synchronous firing is observed after odor detection (Laurent and Davidowitz, 1994). Olfactory information appears to be encoded in the synchronization. Neurons involved in decoding have been identified outside the mushroom body, forming synapses with the intrinsic neurons of the β lobe (McLeod et al., 1998). cAMP signaling pathways in the mushroom body are used for storage of olfactory memory (Skoulakis et al., 1993; Davis and Han, 1996), and short-term memory formation involves α-integrin (Grotewiel et al., 1998).
Pheromones are a major communication channel for insects. For instance, ants use pheromones to identify the colony, signal alarms, mark trails to food, attract workers to brood and to the queen, and bring males and females together for mating (Hölldobler and Wilson, 1990). Queen pheromones also may be involved in the maintenance of polygyny (multiple queen colonies) (Keller and Ross, 1998; Ross and Keller, 1998; Krieger and Ross, 2002) and in founding slave-making colonies (Mori et al., 2000). In addition, foraging, feeding, and defending the nest depend on detection of general odors and tastes and on detection of kairomones (signals from other species). Similarly, many other insects rely upon pheromones, often for similar types of communications.
The isolation and identification of various pheromones from insects is significant for a variety of reasons. For instance, these pheromones would assist in uncovering new basic information about the olfactory communication system and social behavior of social insects. Furthermore, knowledge of the pheromones would have direct application to management and control of various insect pests, including termites and fire ants. Unfortunately, pheromones are present in small quantities in natural sources, and may be difficult to isolate using conventional techniques. A need therefore exists for new methods of isolating pheromones.
SUMMARY OF THE INVENTION
The present invention relates generally to new methods of identifying, isolating, and/or using insect pheromones.
In some embodiments, the invention relates to methods of identifying an insect pheromone comprising: obtaining one or more candidate insect pheromone-binding protein; contacting the candidate pheromone-binding protein with a composition comprising one or more insect pheromones under conditions conducive to allow at least one suitable pheromone, if present, to bind to the candidate pheromone binding protein; eluting any pheromone bound to the isolated candidate pheromone binding protein. These methods may further comprise isolating or purifying the eluted pheromone, using any method known to one of skill in the art. Additionally, the method may further comprise using the isolated pheromone to attract an insect to poison; to repel an insect from an area intended to be kept pest-free; or to interfere with insect behavior, such as mating or foraging, resulting in the extermination of the insect. The present invention is equally applicable to insects in general including, but not limited to, a member of the genus Anopheles, a member of the genus Solenopsis, a member of the genus Aphids, and scale insects, and any other insects that are human, animal and/or plant pests.
In certain exemplary, but not limiting, embodiments, the pheromone-binding protein is from an ant. In more specific non-limiting examples, the ant is a red imported fire ant (Solenopsis invicta). In some cases, the ant is a male ant, worker ant, monogyne queen ant, or polygyne queen ant.
In some cases, the candidate pheromone binding protein is obtained by a process comprising two-dimensional polyarcylamide gel electrophoresis or chromatography.
In some embodiments, obtaining the candidate pheromone binding protein further comprises: obtaining a first sequence analysis of the candidate pheromone-binding protein; and selecting one or more pheromone-binding proteins of interest from the sequenced pheromone-binding proteins. In some cases, the first sequence analysis is performed by mass spectrometry or Edman degradation. The sequence analysis can be either a full or partial sequence analysis. Selecting the pheromone-binding protein of interest, in some non-limiting embodiments, comprises: comparing the first sequence analysis with a second sequence of a known insect pheromone- or odorant-binding protein; and choosing one or more of the sequenced pheromone-binding proteins that have a sequence similar to the second sequence of the known insect pheromone- or odorant-binding protein.
In some preferred embodiments, the candidate pheromone-binding protein is a recombinant pheromone-binding protein. For example, the recombinant pheromone-binding protein may be obtained by a method comprising amplifying a nucleic acid sequence encoding a polypeptide comprising the candidate pheromone-binding protein. Such a method may comprise: obtaining a PCR primer constructed using the candidate pheromone-binding protein; using the PCR primers to amplify a nucleic acid sequence from cDNA produced from mRNA obtained from the insect sample; and expressing the nucleic acid sequence in an expression system to produce the candidate pheromone-binding protein. In some embodiments, the DNA library comprises all or part of an S. invicta genome, an Anopheles genome, an Aphids genome, a scale insect genome, or any other insect genome. The candidate insect pheromones of the composition are extracted, for example, from whole insects, parts of insects, larvae, pupae, or nest middens. Typically, the pheromones are extracted with a solvent, for example, a solvent comprising pentane, hexane, methylene chloride, chloroform, methanol, diethyl ether, or a combination thereof. Further, the composition can be a vapor.
In some specific embodiments, the invention relates to methods of identifying an insect pheromone comprising: obtaining one or more candidate pheromone-binding proteins; performing a sequence analysis of the candidate pheromone-binding proteins; determining a nucleic acid sequence that encodes the candidate pheromone-binding protein; recombinantly expressing the candidate pheromone-binding protein from the nucleic acid sequence; contacting the candidate recombinant pheromone-binding protein with a second composition comprising one or more insect pheromones under conditions conducive to allow at least one suitable pheromone, if present, to bind to the recombinant pheromone-binding protein; and eluting any pheromone that is bound to the recombinant pheromone- or odorant-binding protein. These methods may further comprise isolating or purifying the eluted pheromone. Additionally, the sequence analysis may be either a partial or complete sequence analysis.
In some embodiments of the invention, the candidate recombinant pheromone-binding protein is placed on a solid support prior to the elution step. The solid support may comprise, for example, agarose, plastic, or glass. In some cases, the second composition is a vapor or solvent extract. The method may further comprise using the candidate pheromone-binding protein to construct a primer, and using the primer to identify a genomic sequence from an existing DNA library.
In other embodiments, the invention relates to one or more insect pheromones isolated by the above-discussed methods.
In further embodiments, the invention relates to methods for assaying the specific binding of a pheromone to a pheromone-binding protein, wherein the pheromone is isolated by a process comprising: obtaining one or more candidate insect pheromone-binding proteins; contacting the candidate pheromone-binding protein with a composition comprising one or more insect pheromones under conditions conducive to allow at least one suitable pheromone, if present, to bind to the candidate pheromone-binding protein; eluting the pheromone that is bound to the isolated pheromone-binding proteins; and collecting the eluted pheromone. In specific embodiments, the assay comprises: contacting a sample comprising a pheromone-binding protein with the eluted pheromone under conditions conducive to allow at least one of the eluted pheromone to bind to the pheromone-binding protein of the sample; and measuring the specific binding of the eluted pheromone to the pheromone-binding protein of the sample. For example, the specific binding of the eluted pheromone may be measured by a resonant mirror detector, a plasmon resonance detector, affinity chromatography, a vapor equilibration method, a precipitation method, or a radioligand exchange method.
It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1. Electroblot of two-dimensional polyacrylamide gel electrophoresis of proteins extracted from male fire ant antennae. Horizontal dimension: isoelectric focusing, from pI=6.5 (left) to pI=3.0 (right). Vertical dimension, SDS-PAGE, with molecular weight markers indicated on the left (from bottom: 14.4, 21.5, 31, 45, 66.2, and 97.4 kDa). Blot stained with Coomassie blue and imaged with a CCD camera. Circled spot corresponds to apolipophorin-III.
FIGS. 2A–2B. Illustrates silver staining of worker (FIG. 2A) and male (FIG. 2B) fire ant antennae, showing the sexual dimorphism in sensilla diversity. The stain identifies porous sensilla, which have an olfactory function. b=sensilla basiconica; t=sensilla tricodea. Light micrographs; scale bar=20 μm.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. The Present Invention
The present invention is directed generally to a method of identifying an insect pheromone. Initially, a candidate insect pheromone- or odorant-binding protein is obtained. In the present invention, the general terms “odorant-binding protein” and “pheromone-binding protein” are used to refer to insect proteins that are known as odorant-binding proteins (OBP), pheromone binding protein (PBP), chemosensory protein (CSP), sensory appendage protein (SAP), and also other hydrophobic ligand-binding proteins which may be found in insect antennae or other insect chemosensory organs and which have a biological function to bind to insect pheromones or metabolic precursors or products of insect pheromones. The protein may be obtained in a variety of ways. For instance, the genomic sequences of odorant and pheromone binding proteins obtained from the antennae of an insect may initially be determined. A selected protein may then be used to identify pheromones. For instance, the selected protein may be exposed to a solution comprising multiple pheromones. Any pheromone that is bound to the selected protein may then be eluted and analyzed.
II. OBPs and PBPs
Olfactory sensilla have thin chitin-based cuticle (Steinbrecht and Stankiewicz, 1999) containing small pores through which odors and pheromones may pass. The extracellular fluid inside the sensilla, the antennal lymph, contains high concentrations of odorant-binding proteins (OBPs) or pheromone-binding proteins (PBPs), which capture the odorants or pheromones, respectively (Pelosi and Maida, 1995). Since volatile odorants or pheromones typically are not very soluble in water, the OBPs or PBPs capture and concentrate these molecules. At least one OBP has been shown to be essential for detection of a specific odor (Kim et al., 1998). OBP/PBPs may also be involved in interaction with the olfactory receptors and in removal of odorants from the antennal lymph. Often more than one OBP and PBP occurs in a particular insect species.
The different OBPs or PBPs are localized to particular subgroups of sensilla (Vogt et al., 1991; Pikelny et al., 1994). Some sensilla may contain more than one type of OBP (Hekmat-Scafe et al., 1997). Thus, their localization resembles the distribution of olfactory receptor neurons, which appear to specialize in particular odors, but which may occur together with different types of receptor neurons in the same sensillum. However, the OBPs and PBPs are not as specific in their binding characteristics as the membrane-bound olfactory receptors. For example, two different species of moth, which use two different isomers of the same molecule for the sex pheromone, have the same PBP for both molecules (Willett and Harrison, 1999). Furthermore, a PBP was found to be incapable of distinguishing between the R and S enantiomers of a beetle pheromone, in contrast to the olfactory receptors for this pheromone, which are enantiomer-specific (Wojtasek et al., 1998). The dissociation constants for ligands from PBPs are in the micromolar range (Du and Prestwich, 1995), which is several orders of magnitude weaker than ligand dissociation constants found for G-coupled receptors (Caron and Lefkowitz, 1976), but similar to the interactions found for vertebrate OBPs (Vincent et al., 2000). Ligand release appears to be triggered by low pH, which causes a substantial conformational change in the OBP (Wojtasek and Leal, 1999; Horst et al., 2001). The negative surface charge of the receptor membrane may be sufficient to lower the surface pH to the level which triggers the ligand-releasing conformational change.
Although the insect olfactory system resembles the vertebrate system in broad outline, many details are quite different. For example, the major insect OBPs and PBPs are from a different protein family than the vertebrate OBPs and PBPs. Vertebrate OBPs are lipocalins, a family of hydrophobic ligand binding proteins that includes serum retinol binding protein and orosomucoid (Flower, 1996). Lipocalins have been found in insects but so far not in the antenna (Holden et al., 1987; Korchi et al., 1999; Tsuchihara et al., 2000). The structural differences between lipocalins and insect OBP/PBPs have been proven by X-ray diffraction and NMR (Sandler et al., 2000; Horst et al., 2001). In contrast to the β-barrel structure of lipocalins, the insect PBP folds into a basket of six α-helices surrounding a hydrophobic pocket where the pheromone molecule binds. The insect OBP/PBP sequences all have six conserved cysteines in disulfide bonds (Leal et al., 1999). Proteins related to the insect OBP/PBP family include the B-protein of the tubular accessory gland secretion of Tenibrio molitor (Paesen and Happ, 1995), and also Tenebrio hemolymph protein THP12 (Rothemund et. al., 1999). Although most of the OBP/PBPs involved in insect olfaction appear to belong to this hexahelical protein family, several putative OBPs have been reported with different structures (Mameli et al., 1996; Picimbon and Leal, 1999; Ishida et al., 2002). This alternative odorant- and pheromone-binding protein family is referred to as the chemosensory protein (CSP) or sensory appendage protein (SAP) family.
In Drosophila menalogaster, about 50 different olfactory receptor genes and 14 different OBP/PBP genes have been identified in the complete genome (Rubin et al., 2000). It seems unlikely that many others will be discovered. Thus, the number of different olfactory receptor genes in Drosophila is quite small—a surprising result, considering that the much simpler organism C. elegans has about 1000 different olfactory receptor genes (Rubin et al., 2000). Although there is some speculation that this difference may have to do with an evolutionary advance in signal processing in insects (Pilpel and Lancet, 1999), there could be other explanations. For example, Drosophila may be a specialist in a narrow range of odors, requiring a highly focused olfactory system. Evidence for this comes from studies of the glomeruli. Drosophila has only about 50 glomeruli in each antennal lobe. By contrast, the carpenter ant Camponotus floridanus has around 200 (Gronenberg et al., 1996). Presumably the demands of sociality would require a complex pheromone repertoire and therefore a larger signal processing apparatus. The Drosophila genome also has at least four CSP/SAPs. A second insect genome (Anopheles gambiae) was recently completed. Anopheles has at least 18 OBP/PBPs and 6 CSP/SAPs. No members of the apolipophorin-III (ALP-III) family could be identified in the Drosophila genome.
III. Identification of Pheromones
In one preferred embodiment of the present invention, pheromone-binding proteins and odorant-binding proteins from an insect are initially characterized. Much of the discussion herein focuses on the use of ants as the selected insect. However, one skilled in the art will recognize that the present invention is equally applicable to insects in general including, but not limited to, Anopheles, Aphids, scale insects, and any other insects that are human, animal and/or plant pests.
Pheromone-binding proteins and odorant-binding proteins may be obtained from parts, such as the antennae, of the selected insect. Polyacrylamide gel electrophoresis may be used to purify PBPs and OBPs from pooled antennal segments or other insect parts. Gel bands may be excised, followed by trypsin digestion and mass spectrometric sequence analysis. Alternatively, gels may be electroblotted to medium such as PVDF membrane, and the N-terminal sequence may be determined by automated Edman degradation. The protein sequences may be used to design PCR primers to identify the nucleotide sequences, as well as related sequences, from purified antennal RNA, existing cDNA libraries or BAC libraries of the genome of the selected insect species, such as the S. invicta genome. The sequences may be incorporated into an expression system to produce large quantities of protein. These proteins may then be used to search for and extract scarce ligands. Newly identified candidate pheromones may be tested by electrophysiology and bioassay.
In some embodiments of the invention an amino acid sequence, such as a partial amino acid sequence, may be obtained from a nucleotide sequence using standard molecular biology techniques and the codon table. Additionally, it is possible for one of ordinary skill in the art to use the codon table, and standard molecular biology techniques to obtain a nucleic acid sequence encoding all or part of a known amino acid sequence. For example, as discussed below, SEQ ID NO:1 is a nucleic acid sequence encoding all but the first three amino acids of SEQ ID NO:2. By using standard techniques one may obtain a nucleic acid encoding the entire amino acid sequence of SEQ ID NO:2. For example, one could create a synthetic nucleic acid encoding the first three amino acids and ligate that synthetic nucleic acid onto the native nucleic acid having the sequence of SEQ ID NO:1. Alternatively, one could use all or part of a nucleic acid having the sequence of SEQ ID NO:1 as a probe or primer to PCR a full length native nucleic acid segment encoding the entire amino acid segment of SEQ ID NO:2. Of course, this is only one example of the use of such techniques in the context of the invention. Standard molecular biology techniques are well known to those of skill in the art (see, Sambrook et al., 2000, Maniatis et al., 1990 and Ausbubel et al., 1994, incorporated herein by reference). Table I below list the codons for various species as are well known in the art.







TABLE 1







Codon Table








Amino Acids
Codons













Alanine
Ala
A
GCA GCC GCG GCU


 


Cysteine
Cys
C
UGC UGU


 


Aspartic acid
Asp
D
GAC GAU


 


Glutamic acid
Glu
E
GAA GAG


 


Phenylalanine
Phe
F
UUC UUU


 


Glycine
Gly
G
GGA GGC GGG GGU


 


Histidine
His
H
CAC CAU


 


Isoleucine
Ile
I
AUA AUC AUU


 


Lysine
Lys
K
AAA AAG


 


Leucine
Leu
L
UUA UUG CUA CUC CUG CUU


 


Methionine
Met
M
AUG


 


Asparagine
Asn
N
AAC AAU


 


Proline
Pro
P
CCA CCC CCG CCU


 


Glutamine
Gln
Q
CAA CAG


 


Arginine
Arg
R
AGA AGG CGA CGC CGG CGU


 


Serine
Ser
S
AGC AGU UCA UCC UCG UCU


 


Threonine
Thr
T
ACA ACC ACG ACU


 


Valine
Val
V
GUA GUC GUG GUU


 


Tryptophan
Trp
W
UGG


 


Tyrosine
Tyr
Y
UAC UAU









A. Determination of Partial Amino Acid Sequences of PBP/OBP
Insects that may be used in the present invention may be readily collected and stored using conventional techniques. For instance, if fire ants are used, the ants may be collected by the floatation method (Jouvenaz et al., 1977) and maintained in plastic trays (Hölldobler and Wilson, 1990). The antennae from the insects may then be collected, and placed in a suitable buffer solution to dissolve at least some of the PBPs and OBPs present in the antennae. The proteins may then be separated out of the solution using conventional techniques.
In one particular embodiment, antennae or other parts from the collected insects may be dissected on a freeze tray and, if necessary, subdivided into individual antennomers. Under a dissecting microscope, pooled antennomers may be transferred to a ceramic mortar in Laemmli (1970) SDS sample dilution buffer, or an immobilized pH gradient buffer containing 8 M urea, or other suitable buffer solution. For instance, 100–300 antennae may be transferred to a small ceramic mortar, such as one having a diameter of about 3 cm, in 20 μL of sample buffer. The antennae may then be ground with a pestle to break the cuticle. The extract and fragments may be washed into a centrifuge tube with additional aliquots of the sample buffer. For instance, the 20 μL of buffer solution containing the antennae may be washed into a 1.5 mL plastic centrifuge tube with two additional 20 μL aliquots of sample buffer. After centrifugation, the proteins may be separated by a variety of conventional techniques. For instance, the supernatant may be applied to a 15% polyacrylamide gel and the proteins separated by electrophoresis, or applied to an immobilized pH gradient strip and the proteins separated by isoelectric focusing followed by SDS-PAGE.
N-terminal sequences may be determined by electroblotting gels onto PVDF film (Matsudaira, 1987) followed by gas-phase Edman sequencing. Internal sequences may be obtained as follows. After staining, bands within a specified range, such as 14–23 kDa, may be excised and digested with trypsin, as described by Shevchenko et al. (1996) or other suitable compounds. The tryptic fragments may then be analyzed, such as in a Finnigan LCQ mass spectrometer, using the MS/MS capabilities of this Finnigan instrument to provide sequence information. Sequences obtained may be used to construct PCRTM primers.
B. Determination of Full Genomic Sequences of PBP/OBPs and Related Insect Proteins
Antennae or other insect parts may be obtained by dissection of late stage pupae or adult insects that may be frozen on dry ice. mRNA may be isolated by homogenizing tissue in a suitable solution and then extracting and purifying it. For instance, mRNA may be isolated by homogenizing tissue in TNE:phenol (1:1), extracting proteins with phenol:chloroform 1:1 and purification with an oligo-dT column. A first strand cDNA may then be prepared. The strand may be prepared, for example, using MLV reverse transcriptase and either poly T or random octamer primers. The single strand cDNA may be used in PCRTM reactions with degenerate primers selected from the tryptic peptide sequences to obtain probes. Since OBP/PBPs are small (14–18 kDa), the coding domain of the cDNA should not be difficult to obtain by these methods of PCRTM amplification. Amplified PCRTM products may be ligated into the pGEM TA vector (Promega) and their sequences may be determined to ensure that they encode putative OBP/PBPs. Sequences may be compared with insect OBP/PBP sequences obtained from GenBank, EMBL and other sequence databases using suitable programs, such as Geneworks and BLAST. Isolates may be used to probe genomic libraries at lower stringency to search for additional PBP/OBP genes.
C. Distinguishing PBPs from GOBPs and Related Proteins
In cases where many OBP/PBP candidate sequences are obtained (e.g. from a complete genome), relevant PBP sequences may be distinguished from GOBP sequences by several methods.
1) A PBP involved in a specific behavior will typically be expressed only in those insects displaying the behavior. For example, a PBP that binds to a sex pheromone usually would be expressed exclusively in males or females. Therefore, the protein and the mRNA for it would be found only in males or only in females. This can be determined by gel electrophoresis or Northern blots. Similarly, in social insects, a PBP involved in a caste-specific behavior would be expressed only in a particular caste (e.g. nurses, soldiers or foragers).
2) The anatomical location of OBP/PBP expression in the insect may give clues about function. Some OBP/PBP-like proteins have been found in hemolymph or in secretions, and these proteins may be unrelated to pheromone binding (e.g. the B-proteins and the THP12 protein of Tenebrio molitor). Such OBP/PBP-like proteins can be distinguished from PBPs by their location of expression. In situ hybridization may be used to locate the tissue where a candidate sequence is expressed. PBPs are expected to be primarily expressed in the antenna (although interesting exceptions may be found: for example, Gp-9 in S. invicta is expressed in the thorax).
3) There appear to be some sequence differences between PBPs and OBPs. More than 100 insect PBP/OBP sequences are identified in the Pfam database. Within this set, about 20 different sequences have been identified as PBPs and about 20 as GOBPs. All of the proteins in the insect OBP/PBP family have the distinctive sequence pattern CXXXC. In the PBPs, CXXXC is most often followed by L, whereas in the OBPs, it is most often followed by M, or an amino acid other than L. Furthermore, the C-terminal sequence after the sixth cysteine is, on average, 22 amino acids in PBPs and only 18 amino acids in GOBPs. Therefore, in attempting to distinguish a PBP sequence from a GOBP sequence, one should choose sequences containing CXXXCL and a C-terminal sequence of approximately 22 amino acids.
D. Expression of PBPs in a Recombinant System.
Isolated PBP genes may be inserted into a pET22b plasmid, which permits high yield periplasmic expression of insect pheromone binding proteins in E. coli (Wojtasek and Leal, 1999). With this system, 6–10 mg pure B. mori PBP was produced per liter of culture. Recombinant PBP may be purified by the methods used by Wojtasek and Leal (1999) for B. mori PBP: successive chromatography on DEAE and hydroxyapatite, followed by gel permeation chromatography.
E. Identifying Pheromones
Expression of recombinant PBPs provides the means to identify the pheromones that bind to the proteins. This reverse strategy is known as “ligand fishing” (Catimel et al., 2000). In a typical experiment, a purified PBP is covalently attached to CNBr activated agarose. Extracts may be prepared from whole insects. For instance, with respect to ants, extracts may be prepared from whole ants (different castes, workers with different task assignments), ant cuticle, larvae, pupae, nest middens, dissected segments, and dissected glands. A variety of solvents may be tested for extraction, including, for example pentane, hexane, methylene chloride, chloroform, chloroform/methanol 1:1, and diethyl ether. Those skilled in the art will recognize that a variety of other extracts may also be used.
The extracts may be diluted into aqueous mixtures of solvents expected to be compatible with the native protein structure, such as, for example, dioxane, dimethyl formamide and DMSO. For example, dioxane may be tried initially because the B. mori PBP was crystallized in its native structure from a 50% solution of PEG 20,000 (Sandler et al., 2000), a polyether chemically similar to dioxane. It is preferable to seek a trade-off between high dilution of the extract and relatively low amount of organic solvent, for example about 10–25%, which will combine to keep the pheromones in solution and the protein in its native conformation. Ligands may be eluted from the PBP-agarose matrix by a combined pH-jump to, for example, pH 4.5 and an increase in the organic solvent content. Eluted material may be analyzed by a variety of means, including GC/MS.
Recombinant protein may be covalently attached to a suitable substrate, such as the aminosilane substrate of the Iasys biosensor cuvet (Affinity Sensors, Franklin, Mass.). After exposure to the previously described insect extracts, crude chromatographic fractions, or chromatographically purified components of extracts, specific binding to the PBP may be measured. Specific binding may be measured, for example, by the resonant mirror detector, following procedures similar to Beuckmann et al. (1999). This method may be used both for searching crude extracts for unknown pheromones and other ligands and for measuring equilibrium binding constants of purified candidate pheromones.
If affinity chromatography and the resonant mirror biosensor methods are inconclusive, other techniques may also be tried. One such technique is the vapor equilibration method. Samples of recombinant PBPs may be equilibrated with test vapors of crude pheromone extracts or purified fractions, according to the method used by Briand et al. (2000) to study ligand binding to hamster PBP. Bound ligand may extracted in pentane, methylene chloride, chloroform or other suitable organic solvent, and identified by GC/MS or other means.
Another technique that may be used is the precipitation method. This method, developed by Danty et al. (1999) for studying pheromone binding to Apis PBP, is similar to the vapor equilibration method except that the candidate pheromones are equilibrated with the PBP in the liquid phase and the protein-ligand complexes are precipitated with ammonium sulfate.
A third group of techniques that may be used is radioligand exchange methods. A competitive inhibitor binding assay may be possible if a non-native ligand can be identified which specifically binds to the PBP. The key feature of this inhibitor will be commercial availability with a radionuclide, or ease of incorporating a radionuclide. The inhibitor may be bound to the PBP and then dissociated by varying concentrations of non-labeled candidate pheromone. In an adaptation of the method of Du and Prestwich (1995) to study ligand binding, the pheromone-ligand complex may be removed from solution by binding to a coated plastic surface. The free ligand may be measured by determining the radioactivity remaining in the solution. Alternatively, in an adaptation of the method of Vincent et al. (2000), the displacement of radiolabeled pheromone from the PBP may be measured by vapor diffusion.
IV. EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
Inference of the Presence of a Simple Insect Pheromone System by Anatomical and Behavioral Analysis
This example shows how observation of sexual dimorphism can reveal a simple pheromone system. Observation of whole red imported fire ants by light microscopy (100×) shows differences between male and female antennae. The female antenna has 10 (worker) or 11 (queen) segments, with the distal segments enlarged into a club. The male antenna lacks a club and contains 12 segments. The inventors examined the antennae by scanning electron microscopy. Ants were collected by shoveling the mound into a talc-lined bucket and slowly floating the ants to the surface with water (Jouvanaz et al., 1977). Males were identified by their black cuticle, small head and jaws, and filaform antennae. Ants were fixed overnight in 0.2 M phosphate buffer, pH 7.2 containing 2% glutaraldehyde and 2% paraformaledhyde, and then dehydrated in an alcohol series. Subsequently, the ants were critical point dried, coated with gold, and imaged in a JEOL 840 instrument. Female antennae showed a variety of external sensilla types on the club, including sensilla basiconica and four different types of sensilla tricodea. By contrast, the external sensilla on the male antennae were almost exclusively a single type of sensilla tricodea (FIG. 2). In separate preparations, the antennae were stained with silver to discover which sensilla contained pores (Navasero and Elzen, 1991). Ants were soaked for 5 min in 0.1 M AgNO3. A few drops of Kodak Photoflo, or 10% Triton X-100 were added to keep the ants submerged. After a brief water rinse, the ants were soaked for 5 min in Kodak Microdal-X developer. The ants were then washed in 3% acetic acid and dehydrated in an alcohol series. The antennae were removed and embedded in Cytoseal for light microscopy. The results (FIG. 2) show that various types of porous sensilla occur on the female antenna, nearly all on the club. By contrast, the male antenna is uniformly covered with nearly identical porous sensilla tricodea. These anatomical observations suggest that the male fire ant is sensitive to a limited range of odor and pheromone signals compared to the female. Male fire ants do not care for brood, forage, or maintain and defend the nest. Their sole function in the colony is to participate in nuptial flights. This behavior pattern, combined with the simple sensilla pattern on the antenna, lead to the inference that male fire ants are sensitive to a limited number of pheromones and may have only a few pheromone-binding proteins.
Example 2
Isolation and Amino Acid Sequence Analysis of a Putative Pheromone-Binding Protein from Male Fire Ants
This example demonstrates how a pheromone-binding protein sequence is obtained. Two hundred and fifty male fire ants were collected as described in Example 1. The antennae were removed by dissection on a −20° C. cold plate under a dissecting microscope and stored at −20° C. The antennae were transferred to a small ceramic mortar and, under a dissecting microscope, ground with a pestle in 40 μL of “rehydration buffer” prepared from a mixture of 12 g urea, 125 μL pH 3–10 IPG buffer (Pharmacia), and 16 mL deionized water. The resulting suspension of antenna fragments in buffer was transferred to a 1.5 mL plastic centrifuge tube and the mortar was rinsed with 20 μL of rehydration buffer, which was combined with the antenna fragment suspension in the centrifuge tube. The tube was centrifuged at 4000×g. The supernatant was diluted with 230 μL of rehydration buffer and placed in an Immobiline Dry Strip Reswelling Tray (Pharmacia) along with a 13 cm pH 3–10 Immobiline Dry Strip (Pharmacia).
After rehydration, the strip was subjected to isoelectric focusing at 3500 V for 5 hrs. The strip was then cut to produce a pH 3–6.5 half (“acidic”) and a pH 6.5–10 half (“basic”). The half strips were separately equilbrated for 10 min in 0.5 M Tris buffer, pH 6.8 containing 0.25% dithiothreitol and 10 min in a solution prepared from 2 mL of 0.5 M Tris buffer, pH 6.8, 7.2 g urea, 6 mL glycerol, 0.2 g sodium dodecyl sulfate, 6.7 mL deionized water, and 90 mg iodoacetamide. The strips were then individually applied to the tops of 12% 8.5×6×0.75 cm polyacrylamide gels prepared according to the method of Laemmli (1970) and containing 1 cm stacking gels. Electrophoresis was performed for 45 min at 200 V. The gels were then soaked for 5 min in 3 mM CAPS buffer, pH 11 containing 10% methanol and electroblotted to PVDF film by the method of Matsudaira (1987) (e.g. 1 hr at 100 V in a BioRad Mini Trans-Blot Electrophoretic Transfer Cell). The PVDF films were stained for 1 min with 0.1% Coomassie R250 in 50% methanol and destained for approximately 5 min with a solution of 50% methanol, 10% acetic acid.
Two dimensional gel electrophoresis of extracts from whole male fire ant antennae shows one major spot at low apparent molecular weight and acidic isoelectric point (FIG. 1). This protein is near the range observed for OBP/PBPs in other insects. Further analysis of this protein by automated Edman degradation shows that the N-terminal sequence is TEGEQSGTQPQLS (SEQ ID NO:3). The sequence was used to design degenerate PCRTM primers. A 700 nucleotide RT-PCR product was obtained using these primers with male fire ant antennal RNA. This product was inserted into a pGEM vector and cloned in E. coli. The full DNA sequence of the insert was determined (SEQ ID NO:1). The derived protein sequence contained the expected N-terminal protein sequence (SEQ ID NO:2). A BLAST search indicated the protein as being 23% identical to apolipophorin-III (ALP-III) from Derobrachus geminatus (Smith et al., 1994) and 50% identical to an uncharacterized protein from the Apis mellifera brain EST library (gi:15354591).
ALP-III is known to function as a lipid-binding protein (Narayanaswami and Ryan, 2000). A member of the ALP-III family was found to be expressed in a pheromone-secreting gland of Epiphyas postvittana (Liu et al., 2002). Thus, ALP-III-like proteins can have a pheromone-related function. ALP-III-like proteins should be included in a list of insect antennal pheromone-binding proteins. The actual role of ALP-III in pheromone physiology may be binding of metabolic precursors and break-down products of pheromones, pheromone transport to or removal from the hemolymph, or pheromone binding in the olfactory reception process.
Example 3
Isolation and Amino Acid Sequence Analysis of a Putative Pheromone-Binding Protein from Male Fire Ants Identified as W3
As described in Example 2 above, two hundred and fifty male fire ants were collected. The antennae were removed by dissection on a −20° C. cold plate under a dissecting microscope and stored at −20° C. The antennae were transferred to a small ceramic mortar and, under a dissecting microscope, ground with a pestle in 40 μL of “rehydration buffer” prepared from a mixture of 12 g urea, 125 μL pH 3–10 IPG buffer (Pharmacia), and 16 mL deionized water. The resulting suspension of antenna fragments in buffer was transferred to a 1.5 mL plastic centrifuge tube and the mortar was rinsed with 20 μL of rehydration buffer, which was combined with the antenna fragment suspension in the centrifuge tube. The tube was centrifuged at 4000×g. The supernatant was diluted with 230 μL of rehydration buffer and placed in an Immobiline Dry Strip Reswelling Tray (Pharmacia) along with a 13 cm pH 3–10 Immobiline Dry Strip (Pharmacia).
After rehydration, the strip was subjected to isoelectric focusing at 3500 V for 5 hrs. The strip was then cut to produce a pH 3–6.5 half (“acidic”) and a pH 6.5–10 half (“basic”). The half strips were separately equilbrated for 10 min in 0.5 M Tris buffer, pH 6.8 containing 0.25% dithiothreitol and 10 min in a solution prepared from 2 mL of 0.5 M Tris buffer, pH 6.8, 7.2 g urea, 6 mL glycerol, 0.2 g sodium dodecyl sulfate, 6.7 mL deionized water, and 90 mg iodoacetamide. The strips were then individually applied to the tops of 12% 8.5×6×0.75 cm polyacrylamide gels prepared according to the method of Laemmli (1970) and containing 1 cm stacking gels.
Electrophoresis was performed for 45 min at 200 V. The gels were then soaked for 5 min in 3 mM CAPS buffer, pH 11 containing 10% methanol and electroblotted to PVDF film by the method of Matsudaira (1987) (e.g. 1 hr at 100 V in a BioRad Mini Trans-Blot Electrophoretic Transfer Cell). The PVDF films were stained for 1 min with 0.1% Coomassie R250 in 50% methanol and destained for approximately 5 min with a solution of 50% methanol, 10% acetic acid.
The major protein spot was identified at relative molecular weight 14,000 and pI 3.5, called W3. N-terminal sequence analysis of W3 was performed in a Procise cLC492 gas phase sequencer. The sequence obtained was:
W3 GDLGLYPDEL (SEQ ID NO:4)
This sequence does not correspond to any known protein in the NCBI database, using a BLAST search. However, the sequence LY-D is similar to a sequence near the amino terminus of the CSP identified in the antenna of the Argentine ant (Ishida et al., 2002).
The W3 sequence may be used, as described in Example 2 above, to prepare PCRTM primers to obtain the full DNA and protein sequences from antennal cDNA.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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## Claims

1. A method of identifying an insect pheromone comprising:
a) obtaining one or more insect pheromone-binding proteins;
b) obtaining a composition comprising an extract of whole insects, parts of insects, larvae, pupae, or nest middens, wherein the composition potentially comprises one or more insect pheromones;
c) contacting the one or more pheromone-binding proteins with the composition under conditions conducive to allow at least one suitable pheromone, if present, to bind to at least one of the pheromone binding proteins;
d) eluting any molecule bound to the one or more pheromone binding proteins, wherein any eluted molecule is identified as a pheromone.

2. The method of claim 1, further comprising isolating or purifying the eluted molecule.

3. The method of claim 1, further comprising using the isolated pheromone to attract an insect to poison; to repel an insect from an area intended to be kept pest-free; or to interfere with insect behavior, such as mating or foraging, resulting in the extermination of the insect.

4. The method of claim 1, wherein the insect pheromone-binding protein is obtained from a member of the genus Anopheles, an aphid, a member of the genus Solenopsis, or a scale insect.

5. The method of claim 1, wherein the pheromone-binding protein is from red imported fire ants, such as male ants, worker ants, monogyne queen ants, or polygyne queen ants.

6. The method of claim 1, wherein the pheromone-binding protein is obtained by SDS polyacrylamide gel electrophoresis or chromatography.

7. The method of claim 1, wherein obtaining one or more insect pheromone-binding proteins further comprises:
a) obtaining a first sequence analysis of the one or more pheromone-binding proteins; and
b) selecting one or more pheromone-binding proteins of interest from the sequenced pheromone-binding proteins.

8. The method of claim 7, wherein the first sequence analysis is performed by mass spectrometry or Edman degradation.

9. The method of claim 7, wherein the sequence analysis is a partial sequence analysis.

10. The method of claim 7, wherein selecting the pheromone-binding protein of interest comprises:
a) comparing the first sequence analysis with a second sequence of a known insect pheromone- or odorant-binding protein; and
b) choosing one or more of the sequenced pheromone-binding proteins that have a sequence similar to the second sequence of the known insect pheromone- or odorant-binding protein.

11. The method of claim 1, wherein the pheromone-binding protein is a recombinant pheromone binding protein.

12. The method of claim 1, wherein the composition is a vapor.

13. The method of claim 1, wherein the pheromones are extracted with a solvent.

14. The method of claim 13, wherein the solvent comprises pentane, hexane, methylene chloride, chloroform, methanol, diethyl ether, or a combination thereof.