* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Discovering Pheromones of the Red Imported Fire Ant (Solenopsis
Survey
Document related concepts
Magnesium transporter wikipedia , lookup
Protein (nutrient) wikipedia , lookup
Protein phosphorylation wikipedia , lookup
List of types of proteins wikipedia , lookup
Protein folding wikipedia , lookup
Homology modeling wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Protein moonlighting wikipedia , lookup
Signal transduction wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein structure prediction wikipedia , lookup
Western blot wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Transcript
Discovering Pheromones of the Red Imported Fire Ant (Solenopsis invicta Buren): A Review and Proposed New Target for Pheromone Disruption1 Robert Renthal Department of Biology University of Texas at San Antonio, San Antonio, Texas 78249 USA J. Agric. Urban Entomol. 20(3): 113–121 (July 2003) ABSTRACT A method is described for discovering pheromones of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), using affinity chromatography with the pheromone-binding proteins of the ant. Proteins in the pheromone- and odorant-binding protein family, which are abundant in moths and other insect species, are not apparent in fire ant antennae. Two other types of hydrophobic ligand-binding protein—chemosensory protein, and apolipophorin-III—could be important in fire ant olfaction. These proteins may be useful for identifying pheromones by affinity chromatography. The availability of fire ant pheromones could be a valuable addition to other pest management strategies. KEY WORDS Hymenoptera, Formicidae, Solenopsis invicta, red imported fire ant, pheromones, pheromone-binding proteins, chemosensory proteins, encapsulins, apolipophorin-III 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 & Wilson 1990). Queen pheromones also may be involved in the maintenance of polygyny (Keller & Ross 1998, Ross & Keller 1998, Krieger & 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). Clearly, interference with pheromone-based communication in a fire ant colony would be a useful goal for management of the red imported fire ant (RIFA), Solenopsis invicta Buren (Hymenoptera: Formicidae). Pheromones of some insect species have become useful tools in pest management. Two strategies based on pheromones have been used with varying success: attract and kill, in which a sex pheromone lures insects to insecticide (Charmillot et al. 2000); and mating disruption (Shani 2000), in which a mating pheromone is released at the time of mating to mask directional information contained in concentration gradients. Pheromones of relatively large insects can be identified by simple extraction and chromatographic methods, when the glandular source of the pheromone is known and the behavioral response is clear. Pheromone iden- 1 Accepted for publication 13 February 2004. 113 114 J. Agric. Urban Entomol. Vol. 20, No. 3 (2003) tification technology has been enhanced by advances such as electroantennographic recording as a detector for gas chromatography (Arn et al. 1975), and solid phase microextraction techniques (Jones & Oldham 1999). Although many RIFA pheromones have been identified (Hölldobler & Wilson 1990, Williams et al. 1981, Vander Meer et al. 1980, Rocca et al. 1983a,b, Glancy et al. 1984, Vander Meer et al. 1988, Alonso & Vander Meer 1997, Vargo 1997), pheromones have not yet been useful for control of RIFA or any other pest ant species. This may be the result in part, of the complexity of chemical signaling by ants. Vargo & Hulsey (2000) found multiple glandular sources of an ant pheromone. Another difficulty in exploiting ant pheromones is that some responses to releaser pheromones require primer pheromones, which complicates the process of pheromone discovery. My laboratory has been pursuing a strategy of using molecular biology tools to identify fire ant pheromones by exploiting protein components of the ant’s olfactory system. Insect pheromones are received by sensilla primarily located in the antenna, where they first are captured at the air/water interface by pheromonebinding proteins (Pelosi & Maida 1995; Fig. 1). These proteins are thought to increase the solubility of pheromones in the aqueous sensillar lymph, and they may also be involved in signaling to the pheromone receptors in the olfactory neuron membrane. Purified ant pheromone-binding proteins could be used to capture their pheromone targets from extracts of ants, using the technique known as affinity chromatography. In general, this strategy is far more complicated than traditional pheromone purification methods. If the complete genome of an insect species has been sequenced, then it is straightforward to identify potential pheromone-binding protein sequences. However, even for a species such as RIFA, which lacks a sequenced genome, there may be advantages in attempting to identify pheromones via their binding proteins, as explained in the following sections. Pheromone-Binding Protein Families There are several different pheromone-binding protein families. Although these proteins have unusually high levels of amino acid sequence divergence, the families can be distinguished by characteristic amino acid sequence patterns and three-dimensional structures. The first pheromone/odorant-binding proteins were identified in vertebrates (Pevsner et al. 1988). These are members of the lipocalin family of hydrophobic ligand-binding proteins. Lipocalins, with molecular weights in the range of 25 kDa, have sequences containing three distinctive amino acid sequence motifs, and they fold into a -barrel structure (Flower 1996). Lipocalins are found in insects, for example as a pigment carrier in moth hemolymph (Holden et al. 1987), but their role in insect olfaction seems limited. In a swallowtail butterfly, a lipocalin was found to be involved in carrying lipophilic plant substances to tarsal receptors for locating oviposition sites (Tsuchihara et al. 2000); and in a cockroach, a lipocalin was found in a tergal gland secretion (Korchi et al. 1999). So far, lipocalins have not been identified in insect antennae. Two types of antennal ligand-binding proteins that are unique to insect olfaction have been identified: the pheromone-binding protein/odorant-binding protein (PBP/OBP) family, and the chemosensory (or sensory appendage) protein family. The PBP/OBP family consists of proteins with molecular weights in the 12- to 16-kDa range. The amino acid sequences are extremely divergent, but all contain RENTHAL: Targets for Disruption of Pheromone Reception 115 Fig. 1. Cross-section of antenna (left panel) showing sensilla and olfactory receptor neuron. Blow-up (right panel) shows pheromone-binding proteins capturing pheromones and carrying them to the neuronal membrane surface. a pattern of six cysteines that form three disulfide bonds. The sequence can be described in the Prosite notation (http://www.expasy.org/prosite/) as: C-x(26,35)C-x(3)-C-x(36,43)-C-x(8,14)-C-x(8)-C. The three-dimensional structure of the PBP/OBP proteins is known from X-ray crystallography and nuclear magnetic resonance spectroscopy of the Bombyx mori (Lepidoptera: Bombycidae) sex pheromone-binding protein (Sandler et al. 2000, Horst et al. 2001, Lee et al. 2002). The protein consists of a cluster of six or seven ␣-helices surrounding a hydrophobic cavity where the pheromone binds. PBP/OBPs are commonly expressed in antennal sensilla and also in sensilla on mouth parts. In S. invicta, a PBP/OBP family member is expressed in the thorax of queens (Krieger & Ross 2002). Different PBP/OBPs are localized to particular subgroups of sensilla (Vogt et al. 1991, Pikielny et al. 1994). Some sensilla may contain more than one type of PBP/OBP (Hekmat-Scafe et al. 1997). Thus, their localization resembles the distribution of olfactory receptor neurons, which appear to specialize in particular odors and may occur together in the same sensillum with receptor neurons of different specificity. The binding of pheromones or general odors to PBP/OBPs is usually not as specific as binding to the olfactory receptors on the membranes of the olfactory neurons. For example, two different species of moth, which use two different 116 J. Agric. Urban Entomol. Vol. 20, No. 3 (2003) isomers of the same molecule for the sex pheromone, have the same PBP for both molecules (Willett & 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 enantiomer-specific olfactory receptors for this pheromone (Wojtasek et al. 1998). The dissociation constants for ligands from PBP/OBPs were reported to be in the micromolar range (Du & Prestwich 1995), or 0.1 to 1 M (Campanacci et al. 2001). This is several orders of magnitude weaker than ligand dissociation constants found for G-coupled receptors (Caron & Lefkowitz 1976), but similar to the interactions found for vertebrate pheromone-binding proteins (Vincent et al. 2000). Ligand release appears to be triggered by low pH, which causes a substantial conformational change in the OBP (Wojtasek & 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 ligandreleasing conformational change. Proteins related to the insect PBP/ OBP family include the B-protein of the tubular accessory gland secretion of Tenebrio molitor (Coleoptera: Tenebrionidae) (Paesen & Happ 1995), and also Tenebrio hemolymph protein THP12 (Rothemund et al. 1999). A second type of ligand binding protein involved in insect olfaction is known as the chemosensory protein (CSP) or sensory appendage protein (SAP) family (Mameli et al. 1996, Picimbon & Leal 1999, Ishida et al. 2002). These proteins are expressed in antennae, palps, tarsi, and other tissues. The CSP family has less divergent amino acid sequences than the PBP/OBP and lipocalin families. Members of the CSP family have a conserved pattern of four cysteines, which form two disulfide bonds. The sequence can be summarized in Prosite notation as C-x(6,8)C-x(18,19)-C-x(2)-C. The three-dimensional structure, known from X-ray crystallography of CSP from the moth Mamestra brassicae (Lepidoptera: Noctuidae), shows six ␣-helices with a different spatial arrangement from the PBP/OBP family (Lartigue et al. 2002). Most CSPs are smaller than PBP/OBPs, having molecular weights around 13 kDa. Binding of model compounds indicates ligand dissociation constants in the micromolar range (Lartigue et al. 2002). A protein with a sequence similar to the CSP family was isolated from Drosophila melanogaster (Diptera: Drosophilidae) ejaculatory bulb (Bohbot et al. 1998). Because the PBP/OBPs and the CSPs are found in both olfactory and nonolfactory tissues, Leal (2003) recently suggested using the term “encapsulins” to refer to these proteins. This term provides a category for classification of newly discovered proteins that are similar in structure to olfactory proteins but that do not yet have a known biological function. The encapsulins contain two protein families with unrelated amino acid sequences. I suggest using the terms “2X encapsulins” to denote the CSP family and “3X encapsulins” to denote the PBP/ OBP family. The 2X and 3X designations refer to the distinctive sequence patterns in the two families: CXXC in the CSP family, and CXXXC in the PBP/OBP family (using the amino acid single letter code, where C is cysteine and X is any amino acid). Pheromone Discovery When a receptor is known but the ligand molecules that bind to it are unknown, affinity chromatography can be used to identify the ligands. To identify unknown pheromones, a purified pheromone-binding protein can be attached to a RENTHAL: Targets for Disruption of Pheromone Reception 117 solid support, such as agarose (Fig. 2A). An extract containing the pheromones is passed down a chromatography column packed with the agarose-coupled pheromone-binding protein. Unbound molecules are washed away (Fig. 2B), leaving the ligands bound to the column. The ligands can be eluted (e.g. with a pH change, or solvent change) and analyzed by mass spectrometry. Using the pheromonebinding proteins to capture the ligands has the virtue of collecting groups of isomers, in cases where the pheromone is a complex mixture, since the binding specificity is not as exact as the neuronal olfactory receptors (Campanacci et al. 2001). Although the total numbers of pheromone-binding proteins is not known in any ant species, a BLAST search of the Drosophila genome shows at least twelve 3X encapsulin genes and four 2X encapsulin genes, and the Anopheles gambiae (Diptera: Culicidae) genome shows at least eighteen 3X encapsulin genes and six 2X encapsulin genes. This contrasts with about 80 olfactory receptors each in Drosophila and Anopheles. Because of the high sequence diversity of the encapsulins, it is not possible to isolate orthologs from ants using these dipteran sequences to design PCR primers. Instead, it is necessary to isolate the proteins from the ant antenna to obtain an amino terminal amino acid sequence for designing PCR primers. The full-length sequence is then obtained by PCR from antennal cDNA. For subsequent ligand-binding studies, the protein is expressed in bacteria using recombinant DNA techniques. Unfortunately, the RIFA worker antenna shows a large diversity of low molecular weight proteins, making identification of encapsulins difficult. Of the major low molecular weight proteins with free amino termini, one has an N-terminal sequence similar to a soft cuticle protein known in other insect species, and the other may be similar to a CSP first identified in the Argentine ant, Linepithema humile (Hymenoptera: Formicidae) (Ishida et al. 2002). We thought that the RIFA male would have a simpler set of olfactory proteins in the antenna because males have a simpler behavioral repertoire. RIFA male’s only apparent task is to participate in mating flights. The male antennal morphology is much simpler than that of workers (Renthal et al. 2003), suggesting simpler olfactory processing. To our surprise, we found that the major low molecular weight male antennal protein with a free amino terminus is apolipophorin-III (Guntur et al. 2004). This hemolymph protein was first identified in locusts as a lipid transport protein (Narayanaswami & Ryan 2000). Apolipophorin-III (ALP-III) has a molecular weight of 18 kDa and the amino acid sequences from different species are highly divergent. A single distinguishing sequence pattern has not yet been identified. The three-dimensional structures of moth (Wang et al. 1997) and locust (Breiter et al. 1991) ALP-III show a cluster of five parallel ␣-helices, which are thought to open with a hinge-like motion to bind to hemolymph lipophorins I and II. In addition to its function in lipid transport, ALP-III has been found to be involved in immediate immunity (Wiesner et al. 1997), apoptosis (Sun et al. 1995), and pheromone secretion (Liu et al. 2000). Since ALP-III can reversibly bind hydrophobic molecules and is expressed in the male fire ant antenna, we plan to further evaluate this protein for a possible role in pheromone reception. Conclusions In insect species with scarce or heterogeneous encapsulins, it may be possible to purify candidate pheromones by affinity chromatography using proteins ex- 118 J. Agric. Urban Entomol. Vol. 20, No. 3 (2003) Fig. 2. Pheromone-binding protein (PBP) attached to agarose beads. A. Insect extract containing pheromone passed down a column of PBP-agarose. B. Most of extract washes away, leaving pheromone bound to PBP on agarose beads. pressed in bacteria from recombinant DNA sequences. In addition, other hydrophobic ligand binding proteins such as apolipophorin-III, which may be involved in pheromone transport, could also be used for pheromone discovery. Acknowledgment Supported by a grant from the Texas Imported Fire Ant Research and Management Project. References Cited Alonso, L. E. & R. K. Vander Meer. 1997. Source of alate excitant pheromones in the red imported fire ant Solenopsis invicta (Hymenoptera. Formicidae. J. Insect Behav. 10: 541–555. Arn, H., E. Städler & S. Rauscher. 1975. The electroantennographic detector—a selective and sensitive tool in the gas chromatographic analysis of insect pheromones. Z. Naturforsch. C 30: 722–725. Bohbot, J., F. Sobrio, P. Lucas & P. Nagnan-Le Meillour. 1998. Functional characterization of a new class of odorant-binding proteins in the moth Mamestra brassicae. Biochem. Biophys. Res. Commun. 253: 489–494. Breiter, D. R., M. R. Kanost, M. M. Benning, G. Wesenberg, J. H. Law, M. A. Wells, RENTHAL: Targets for Disruption of Pheromone Reception 119 I. Rayment & H. M. Holden. 1991. Molecular structure of an apolipoprotein determined at 2.5 A resolution. Biochemistry 30: 603–608. Campanacci, V., J. Krieger, S. Bette, J. N. Sturgis, A. Lartigue, C. Cambillau, H. Breer & M. Tegoni. 2001. Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay. J. Biol. Chem. 276: 20078–20084. Caron, M. G. & R. J. Lefkowitz. 1976. Solubilization and characterization of the betaadrenergic receptor binding sites of frog erythrocytes. J. Biol. Chem. 251: 2374–2384. Charmillot, P.-J., D. Hofer & D. Pasquier. 2000. Attract and kill: a new method for control of the codling moth Cydia pomonella. Entomol. Exp. Appl. 94: 211–216. Du, G. & G. D. Prestwich. 1995. Protein structure encodes the ligand binding specificity in pheromone binding proteins. Biochemistry 34: 8726–8732. Flower, D. R. 1996. The lipocalin protein family: structure and function. Biochem. J. 318: 1–14. Glancy, B. M., J. Rocca, C. S. Lofgren & J. Tumlinson. 1984. Field tests with synthetic components of the queen recognition pheromone of the red imported fire ant Solenopsis invicta. Sociobiology 9: 19–30. Guntur, K. V. P., D. Velasquez, L. Chadwell, C. A. Carroll, S. T. Weintraub, J. A. Cassill & R. Renthal. 2004. Apolipophorin-III-like protein expressed in the antenna of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae). Arch. Insect Biochem. Physiol., in press. Hekmat-Scafe, D. S., R. A. Steinbrecht & J. R. Carlson. 1997. Coexpression of two odorant-binding protein homologs in Drosophila: implications for olfactory coding. J. Neurosci. 17: 1616–1624. Holden, H. M., W. R. Rypniewski, J. H. Law & I. Rayment. 1987. The molecular structure of insecticyanin from the tobacco hornworm Manduca sexta L. at 2.6 A resolution. EMBO J. 6: 1565–1570. Hölldobler, B. & E. O. Wilson. 1990. The Ants. Belknap Press, Cambridge, Massachusetts. pp. 227–297 Horst, R., F. Damberger, P. Luginbühl, P. Güntert, G. Peng, L. Nikonova, W. S. Leal & K. Wüthrich. 2001. NMR structure reveals intramolecular regulation mechanism for pheromone binding and release. Proc. Nat. Acad. Sci. USA 98: 14374–14379. Ishida, Y., V. Chiang & W. S. Leal. 2002. Protein that makes sense in the Argentine ant. Naturwiss. 89: 505–507. Jones, G. & N. Oldham. 1999. Pheromone analysis using capillary gas chromatographic techniques. J. Chrom. A. 843: 199–236. Keller, L. & K. G. Ross. 1998. Selfish genes: a green beard in the red fire ant. Nature 394: 573–575. Korchi, A., R. Brossut, H. Bouhin & J. Delachambre. 1999. cDNA cloning of an adult male putative lipocalin specific to tergal gland aphrodisiac secretion in an insect (Leucophaea maderae). FEBS Lett. 449: 125–128. Krieger, M. J. B. & K. G. Ross. 2002. Identification of a major gene regulation complex social behavior. Science 295: 328–332. Lartigue, A., V. Campanacci, A. Roussel, A. M. Larsson, T. A. Jones, M. Tegoni & C. Cambillau. 2002. X-ray structure and ligand binding study of a moth chemosensory protein. J. Biol. Chem. 277: 32094–32098. Leal, W. S. 2003. Proteins that make sense, pp. 447–476. In G. Blomquist & R. Vogt [Eds.], Insect pheromone biochemistry and molecular biology. Elsevier Academic Press, London, United Kingdom. Lee, D., F. F. Damberger, G. Peng, R. Horst, P. Güntert, L. Nikonova, W. S. Leal & K. Wüthrich. 2002. NMR structure of the unliganded Bombyx mori pheromone-binding protein at physiological pH. FEBS Lett. 531: 314–318. Liu, W., H. Jiao & W. L. Roelofs. 2000. Genbank sequence gb AAG34698. 120 J. Agric. Urban Entomol. Vol. 20, No. 3 (2003) Mameli, M., A. Tuccini, M. Mazza, R. Petacchi & P. Pelosi. 1996. Soluble proteins in chemosensory organs of phasmids. Insect Biochem. Mol. Biol. 26: 875–882. Mori, A., D. A. Grasso, R. Visicchio & F. Le Moli. 2000. Colony founding in Polyergus rufescens: the role of the Dufor’s gland. Insectes Sociaux 47: 7–10. Narayanaswami, V. & R. O. Ryan. 2000. Molecular basis of exchangeable apolipoprotein function. Biochim. Biophys. Acta 1483: 15–36. Paesen, G. C. & G. Happ. 1995. The B proteins secreted by the tubular accessory sex glands of the male mealworm beetle, Tenebrio molitor, have sequence similarity to moth pheromone-binding proteins. Insect Biochem. Mol. Biol. 25: 401–408. Pelosi, P. & R. Maida. 1995. Odorant-binding proteins in insects. Comp. Biochem. Physiol. 111B: 503–514. Pevsner, J., R. R. Reed, P. G. Feinstein & S. H. Snyder. 1988. Molecular cloning of odorant-binding protein member of a ligand carrier family. Science 241: 336–339. Picimbon, J.-F. & W. S. Leal. 1999. Olfactory soluble proteins of cockroaches. Insect Biochem. Mol. Biol. 29: 973–978. Pikielny, C. W., G. Hasan, F. Rouyer & M. Rosbash. 1994. Members of a family of Drosophila putative odorant-binding proteins are expressed in different subsets of olfactory hairs. Neuron 12: 35–49. Renthal, R., D. Velasquez, D. Olmos, J. Hampton & W. P. Wergin. 2003. Structure and distribution of antennal sensilla of the red imported fire ant. Micron 34:405–413. Rocca, J. R., J. H. Tumlinson, B. M. Glancey & C. S. Lofgren. 1983a. The queen recognition pheromone of Solenopsis invicta: preparation of (E)-6-(1-pentenyl)-2Hpyran-2-one. Tetrahedron Lett. 24: 1889–1892. Rocca, J. R., J. H. Tumlinson, B. M. Glancey & C. S. Lofgren. 1983b. Synthesis and stereochemistry of tetrahydro-3,5-dimethyl-6-(1-methylbutyl)-2H-pyran-2-one, a component of the queen recognition pheromone of Solenopsis invicta. Tetrahedron Lett. 24: 1893–1896. Ross, K. G. & L. Keller. 1998. Genetic control of social organization in an ant. Proc. Nat. Acad. Sci. USA 95: 14232–14237. Rothemund, S., Y.-C. Liou, P. L. Davies, E. Krause & F. Sonnichsen. 1999. A new class of hexahelical insect proteins revealed as carriers of small hydrophobic ligands. Structure 7: 1325–1332. Sandler, B. H., L. Nikonova, W. S. Leal & J. Clardy. 2000. Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein–bombykol complex. Chem. Biol. 7: 143–151. Shani, A. 2000. Chemical communication agents (pheromones) in integrated pest management. Drug Dev. Res. 50: 400–405. Sun, D., R. Ziegler, C. Milligan, S. Fahrbach & L. M. Schwartz. 1995. Apolipophorin III is dramatically up-regulated during the programmed death of insect skeletal muscle and neurons. J. Neurobiol. 26: 119–129. Tsuchihara, K., K. Ueno, A. Yamanaka, K. Isono, K. Endo, R. Nishida, K. Yoshihara & F. Tokunaga. 2000. A putative binding protein for lipophilic substances related to butterfly oviposition. FEBS Lett. 478: 299–303. Vander Meer, R. K., F. Alvarez & C. S. Lofgren. 1988. Isolation of the trail recruitment pheromone of Solenopsis invicta. J. Chem. Ecol. 14:825–838. Vander Meer, R. K., B. M. Glancey, C. S. Lofgren, A. Glover, J. H. Tumlinson & J. Rocca. 1980. The poison sac of red imported fire ant queens: source of a pheromone attractant. Ann. Entomol. Soc. Amer. 73: 609–612. Vargo, E. L. 1997. Poison gland of queen fire ants (Solenopsis invicta) is the source of a primer pheromone. Naturwiss. 84: 507–510. Vargo, E. L. & C. D. Hulsey. 2000. Multiple glandular origins of queen pheromones in the fire ant Solenopsis invicta. J. Insect Physiol. 46: 1151–1159. Vincent, F., S. Spinelli, R. Ramoni, S. Grolli, P. Pelosi, C. Cambillau & M. Tegoni. RENTHAL: Targets for Disruption of Pheromone Reception 121 2000. Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes. J. Mol. Biol. 300: 127–139. Vogt, R. G., G. D. Prestwich & M. R. Lerner. 1991. Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects. J. Neurobiology 22: 74–84. Wang, J., S. M. Gagne, B. D. Sykes & R. O. Ryan. 1997. Insight into lipid surface recognition and reversible conformational adaptations of an exchangeable apolipoprotein by multidimensional NMR techniques. J. Biol. Chem. 272: 17912–17920. Wiesner, A., S. Losen, P. Kopacek, C. Weise & P. Gotz. 1997. Isolated apolipophorin III from Galleria mellonella stimulates the immune reactions of this insect. J. Insect Physiol. 43: 383–391. Willett, C. S. & R. G. Harrison. 1999. Pheromone binding proteins in the European and Asian corn borers: no protein change associated with pheromone differences. Insect Biochem. Mol. Biol. 29: 277–284. Williams, H. J., M. R. Strand & S. B. Vinson. 1981. Trail pheromone of the red imported fire ant Solenopsis invicta (Buren). Experientia 37: 1159–1160. Wojtasek, H., B. S. Hansson & W. S. Leal. 1998. Attracted or repelled?—a matter of two neurons, one pheromone binding protein, and a chiral center. Biochem. Biophys. Res. Commun. 250: 217–222. Wojtasek, H. & W. S. Leal. 1999. Conformational change in the pheromone-binding protein from Bombyx mori induced by pH and by interaction with membranes. J. Biol. Chem. 274: 30950–30956.