W.S. Leal

Department of Entomology, University of California, Davis
CA 95616, USA

Chemical communication involves the production and release of specific chemicals (semiochemicals) by the emitter, and the detection and olfactory processing of these signals leading to appropriate behavioral responses in the receiver (Roelofs, 1995). Chemical attraction is the major means of sexual recruitment in scarab beetles, in particular, rutelines and melolonthines. Females are normally the emitters and males the receivers, and in this case, the semiochemicals are referred to as sex pheromones. Male-released aggregation pheromones have also been reported for a few Dynastinae. Although a few studies have reported the chemical ecology of the dung beetles (Scarabaeinae), most of the emphasis by research programs on chemical communication in scarab beetles has focused on the subfamilies Cetoniinae, Melolonthinae, Dynastinae, and Rutelinae because of their economic importance as agricultural and/or turf pests. Largely, these research projects are aimed at the development of attractants (pheromones or food-type lure compounds) for possible applications in management programs. In my laboratory, we have taken a comprehensive approach to chemical communication in order to gain a better understanding of both the emitters and receivers and pave the way for the development of environmentally sound control strategies. On the one hand, we focused on the chemistry of the emitters (identification and synthesis of pheromones) and studied the biology, biosynthesis and physiology of pheromone production. On the other hand, we investigated the molecular mechanisms of the olfactory processing in the receivers.

PHEROMONE CHEMISTRY

Recent studies have led to the identification of the sex pheromones of various species in the subfamily Rutelinae and Melolonthinae (Leal, 1998). In general, the pheromones of the former are fatty-acid derived compounds, whereas the latter utilizes phenolic, terpenoid, and amino acid derived compounds. Two interesting exceptions to this general rule are the pheromones of Heptophylla picea and Phyllopertha diversa . Although belonging to the Melolonthinae, H. picea utilizes (R,Z)-7,15-hexadecadien- 4-olide (Leal et al., 1996), most likely biosynthesized from stearic acid. On the other hand, P. diversa (Rutelinae) produces an intriguing alkaloid pheromone, which also has medicinal properties (Leal et al., 1997). Utilizing pheromone blends that consist of just a few semiochemicals or even a single constituent, closely related species have attained isolated chemical communication channels and reproductive isolation (Leal, 1999a; 1999b). Species that have the same pheromone system are isolated either temporarily or geographically. Interestingly, Anomala osakana and Popillia japonica utilize enantiomers of a chiral pheromone (japonilure), with one stereoisomer being an attractant and the other a behavioral antagonist. P. japonica and A. osakana produce (R)- and (S)-japonilure, respectively (Tumlinson et al. 1977; Leal, 1996). The pheromone of one species is a behavioral antagonist for the other. It seems that this agonist-anatagonist activities of the enantiomeric pheromones have evolved as part of the isolation mechanism between these two species that share the same habitats in Japan. In general, scarab beetles can detect only the enantiomer produced by the conspecific females, but P. japonica and A. osakana have evolved the ability to detect both enantiomers, one as an attractant and the other as a behavioral antagonist (stop signal).

PHEROMONE BIOLOGY

Pheromone gland cells in A. cuprea females were identified as modified integumental epithelia of the terminal abdominal sclerites (Tada and Leal, 1997). The gland cells are composed of round pheromone secretory cells with canal structures bearing an end apparatus. On the other hand, we determined that in Holotrichia parallela the pheromone is produced in the posterior part of a ball-shaped sac exposed during female calling. Light microscope observation of the posterior part of the gland revealed a cuticular epithelium layer composed of columnar cells, which was assigned as the tissue involved in the pheromone production (Kim and Leal, 1999).

PHEROMONE BIOSYNTHESIS AND PHEROMONE REGULATION

A typical structure of the sex pheromone of rutelines is the five-membered gamma-lactones having a long unsaturated hydrocarbon chain, such as (R,Z)-5-(—)-(oct-1-enyl)oxacyclopentan-2-one (buibuilactone) and (R,Z)-5-(—)-(dec-1-enyl) oxacyclopentan-2-one (japonilure), which are pheromones for a number of species. Using deuterated precursors, it has been demonstrated that the biosynthesis of these compounds starts from saturated fatty acids (palmitic and stearic acid), involves their desaturation followed by stereospecific 8-hydroxylation, chain shortening and cyclization (Leal et al., 1999). Various scarab species have developed pathways to produce unique pheromone molecules by changing either stereospecificity or regiospecificity of the hydroxylation step. Anomala cuprea and Popillia japonica utilize the (R)-8-hydroxylase, whereas the hydroxyylase of A. osakana is specific to the (S)-substrate. It seems that A. rufocuprea is devoid of the enzyme so it makes methyl Z-(5)-tetradecenoate (Tamaki et al., 1985). Pheromone biosynthesis in scarabs is regulated by a PBAN-like factor. The pheromone titer in the glands of decapitated females dramatically decreased 24 hr after surgery, but it resumed after injection of the brain extracts from virgin females. The activity of the brain extracts is lost after treatment with proteinase K. Because BmPBAN is also active, characterization of the gene encoding the peptide was pursued by library screening and PCR. Hitherto, none of the molecular approaches led to the identification of the PBAN gene in scarab beetles. On the other hand, a bioassay-oriented strategy lead to isolation of the active peaks by reversed phase HPLC and ion-exchange chromatography. The small amount of the isolated peptide prevented any further characterization.

MOLECULAR BASIS OF OLFACTION

For their survival, insects heavily depend on their ability to detect chemical signals from the environment, which are buried in complex mixture of odors from a myriad of sources. This has been highlighted in the literature by their highly sensitive and selective olfactory systems for the detection of sex pheromones, particularly in Lepidoptera, which approach the theoretical limit for a detector. While minimal structural modifications to pheromone molecules render them inactive (Kaissling, 1987), a single molecule of the native ligand is reported to be sufficient to activate the pheromone-specific olfactory neurons in the antennae of the silkworm moth, Bombyx mori (Kaissling and Priesner, 1970). There is growing evidence in the literature that this inordinate sensitivity is achieved by a combination of the roles of various olfactory specific proteins, including odorant receptors, odorant-binding proteins, and odorant-degrading enzymes. In order to gain a better understanding of the molecular basis of olfaction, we aimed at identifying and characterizing the pheromone-degrading enzymes, studying the neurophysiological details of pheromone perception “in vivo,” and isolating, identifying, and cloning the genes encoding the pheromone- and odorant-binding proteins. In order to elucidate the function(s) of these proteins, we have been conducting structural studies in collaboration with Jon Clardy (Cornell University) and Kurt Wuthrich (ETH-Switzerland).

PHEROMONE-DEGRADING ENZYMES

Antennal proteins from the extracts of several species of scarab beetles can degrade buibuilactone and japonilure, even those from species that do not use this group of compounds as their pheromones. In some cases there was only one metabolite, identified as the corresponding hydroxy fatty acid. It seems that the deactivation of the lactone signal is obtained by the opening of the lactone ring. Some species, however, degraded the pheromone into several more products. The esterase from A. octiescostata showed significant preference for (R)-japonilure over that of the (S)-enantiomer. This observation is consistent with the fact that this species produces only the (R)-enantiomers of the two pheromone components and it is anosmic to the (S)-lactones. Analysis of the degradation products of the unique pheromone from P. diversa revealed that only the antennal extract of this species can degrade the pheromone. The antennal extracts from 10 other scarab species and 4 lepidoptearn species produced no activity at all. Separation of the antennal extracts showed that the enzymatic activity was associated with the membrane fractions in the absence of cytosol. Analysis of the degradation reaction suggested that the major degradation product was due to a demethylation at the N-1 position; the second product was due to hydroxylation of the aromatic ring. Studies on the degradation along with potential cofactors or inhibitors showed that the enzymatic system requires NADPH and NADH for activity. On the other hand, the enzymatic activity was inhibited by proadifen and metyrapone, two general widely used inhibitors for cytochrome P450 (Wojtasek and Leal, 1999).

DEGRADATION OF PHEROMONES “IN VIVO”

The discovery of the unique pheromone-degrading enzyme in P. diversa and the identification of enzymatic inhibitors opened the way to study pheromone inactivation “in vivo.” When metyrapone was introduced by diffusion into the pheromone-specific sensilla in the antennae of P. diversa, the pheromone detectors became “silent” to lower concentrations after application of a large concentration of the pheromone. The effect of the inhibitor is remarkably different from adaptation as will be discussed later. In addition, metyrapone treatment had no effect on the sennsila of P. diversa tuned to (Z)-3-hexenyl acetate nor did it affect the pheromone-detecting systems in P. japonica, for which pheromone inactivation is achieved with a sensillar esterase.

IDENTIFICATION AND CLONING OF OBPs

We have identified, cloned, and characterized the odorant-binding proteins from a number of scarab species. It is now clear that scarab beetles possess two families of odorant-binding proteins, one with 116 and the other with 133 amino acids, which we named OBP1 and OBP2, respectively. While OBP1 is well conserved among all species of scarab beetles, OBP2 belongs to a more diverse group and, in contrast to OBP1, it has not been detected in all species. Interestingly, OBP2 possesses isoforms, which can be separated by native gel electrophoresis. These isoforms have different binding affinities. For example, one isoform of OBP2 from P. diversa binds bombykol, whereas the other conformation binds japonilure (Wojtasek et al., 1999). Microheterogeneity of the OBPs in scarab beetles is not derived from different gene products, but it is due to the conformational flexibility of the proteins. Consistently, we found only one gene encoding OBP2 in various species..Plenary Lectures Walter Leal ABSTRACT BOOK I – XXI-International Congress of Entomology, Brazil, August 20-26, 2000 XVI Interestingly, in both A. osakana and P. japonica, we could detect only one PBP in the antennal extracts; the proteins from the two species showed a 96% similarity. Due to the limited sensitivity of the detection methods, one cannot rule out the possibility of the presence of proteins expressed at low levels. However, electrophysiological experiments suggest that if two PBPs were involved in the signal transduction of the enantiomers of japonilure they would be expressed at nearly the same level. Single sensillum recordings from the antennae of the Japanese and Osaka beetles showed that enantiospecific receptor neurons respond equally to (R)- and (S)-japonilure. These findings and the observation that a single PBP from A. osakana bound both enantiomers of japonilure apparently with the same affinity suggested that in the antennae of these species, the same PBP may recognize both the pheromone and the “stop signal”, i.e., the enantiomers of japonilure (Wojtasek et al., 1998).

STRUCTURAL BIOLOGY AND FUNCTION OF PBPs

We envisaged that in order to determine the molecular basis of insect olfaction and elucidate the function of PBPs, we needed to study the three-dimensional structure of the pheromone-binding proteins and its interaction with ligands. We embarked in collaborations with two groups (Jon Clardy and Kurt Wuthrich) to determine the 3D crystal and solution structures of the pheromone-binding protein from Bombyx mori. Functional expression of BmPBP was achieved in E. coli periplasm. The protein appeared as a single band in gel electrophoresis and it was homgeneous in most chromatographic systems. However, NMR experiments conducted by the Wuthrich group indicated the existence of at least two conformations at pH 6.2. Throughout the analysis of both the native and recombinant proteins, a remarkable feature of the PBPs appeared. These proteins have dynamic structures, altering their conformations in pH-dependent ways. Studies with model membranes suggested that upon an interaction with the dendritic membrane, PBPs undergo a conformational change that may lead to the release of the pheromone ligand (Wojtasek and Leal, 1999). The three-dimensional structure of the BmPBP with bound bombykol has been determined by X-ray diffraction (Sandler et al., 2000). BmPBP has six helices, and bombykol binds in a completely enclosed hydrophobic cavity formed by four antiparallel helices. Bomkykol is bound in this cavity through numerous hydrophobic interactions. It has been suggested that a pH drop would result in protonation of the histidine residues that form the base of a flexible loop and protonated histidines could destabilize the loop covering the binding pocket. Although the crystal structure did not show clear evidence for dimers, a comprehensive study (Western immunoblotting experiments, mass spectral analysis, gel filtration estimation of molecular masses, and cross-linking reactions), showed that BmPBP is a monomer at acid pH and a dimer at basic, neutral, and slightly acid pH. This suggests that the physiologically relevant pH for the early olfactory processing is not only that of the sensillar lymph (bulk pH), but also the pH at the surface of the dendrides (localized pH) (Leal, 2000).

ACKNOWLEDGMENTS

I gratefully acknowledge the great contribution that my past and present collaborators and members of my research group made to this work. My research projects in Japan were financially supported by a special coordination fund for promoting science and technology by the Science and Technology Agency of Japan and by the Programe for Promotion of Basic Research Activities for Innovative Biosciences (BRAIN). Work in the US was made possible through direct financial support from the department, college, and Chancellors office at UCD.

REFERENCES

Kaissling, K.-E. 1987. pp. 28-32. In K. Colbow [ed.]. R. H. Wright lectures on insect olfaction, Simon Fraser University.

Kaissling, K. E. and Priesner, E. 1970. Die Riechschwelle des Seidenspinners. Naturwissenschaften 57: 23-28.

Kim, J.-Y. and Leal, W. S. 1999. Eversible pheromone gland in a melolonthine beetle, Holotrichia parallela. J. Chem. Ecol. 25: 825-833.

Leal, W. S. 1996. Chemical communication in scarab beetles: Reciprocal behavioral agonist-antagonist activities of chiral pheromones. Proc. Natl. Acad. Sci. U.S.A. 93: 12112-12115.

Leal, W.S. 1998. Chemical Ecology of Phytophagous Scarb Beetles. Annu. Rev. Entomol. 43: 39-61.

Leal, W. S. 1999a. Scarab beetles, pp. 51-68. In J. Hardie, and A. K. Minks [eds.], Pheromones of non-lepidopteran insects associated with agricultural plants. CABI Publishing, NY.

Leal, W. S. 1999b. Mechanisms of chemical communication in scarab beetles, pp. 464-478. In T, Hidaka, Y. Matsumoto, K. Honda, H. Honda, and K. Tatsuki [eds.], Mechanisms of chemical communication in scarab beetles. University of Tokyo Press, Tokyo.

Leal, W. S. 2000. Duality monomer-dimer of the pheromone-binding protein from Bombyx mori. Biochem. Biophysic Res. Commun. 268: 521-529.

Leal, W. S., Kuwahara, S., Ono, M. and Kubota, S. 1996. (R,Z)-7,15-Hexadecadie-4-olide, Sex Pheromone of the Yellowish Elongate Chafer, Heptophylla picea. Bioorg. Med. Chem. 4: 315-321.

Leal, W.S., Zarbin, P.H.G., Wojtasek, H. and Ferreira, J. T. (1999). Biosynthesis of scarab beetle pheromones: Enantioselective 8-hydroxylation of fatty acids. Eur. J. Biochem. 259: 175-180.

Leal, W. S., Zarbin, P. H. G., Wojtasek, H., Kuwahara, S., Hasegawa, M. and Ueda, Y. 1997. Medicinal alkaloid is a sex pheromone. Nature 385: 213.

Roelofs, W, L. 1995. The chemistry of sex attraction, pp. 103-117. In T. Eisner, and J. Meinwald [eds.], Chemical ecology: The chemistry of biotic interaction. National Academy Press, Washington, D. C.

Sandler, B. H., Nikonova, L., Leal, W. S. and Clardy, J. 2000. Sexual attraction in the silkworm moth: structure of the pheromone-binding protein-bombykol complex. Chemistry & Biol. 7: 143-151.

Tada, S., and Leal, W. S. 1997. Localization and morphology of the sex pheromone glands in scarab beetles (Coleoptera: Rutelinae: Melolonthinae). J. Chem. Ecol. 23: 903-915.

Tamaki, Y, Sugie, H, and Noguchi, H. 1985. Methyl (Z)-5-tetradecenoate: sex attractant pheromone of the soybean beetle, Anomala rufocuprea Motschulsky (Coleoptera: Scarabaeidae). Appl. Entomol. Zool. 20: 359-361.

Tumlinson, J. H., Klein, M. G., Doolitle, R. E., and Proveaux, A. T. 1977. Identification of the female Japanese beetle sex pheromone: inhibition of male response by an enantiomer. Science 197: 789-792.

Wojtasek, H., Hansson, B. S. and Leal, W. S. 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. and Leal, W. S. 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.

Wojtasek, H. and Leal, W. S. 1999. Degradation of an alkaloid pheromone from the pale-brown chafer, Phyllopertha diversa (Coleoptera: Scarabeidae), by an insect olfactory cytochrome P450. FEBS Lett. 458: 333-336.

Wojtasek, H., Picimbon, J.-F. and Leal, W. S. 1999. Identification and cloning of odorant binding proteins from the scarab beetle Phyllopertha diversa. Biochem. Biophys. Res. Commun. 263: 832-837.

Index terms: pheromones, pheromone-binding proteins, pheromone-degrading enzymes, biosynthesis

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