Chem. Senses 26: 49-54,
2001
© Oxford University Press 2001
A Photoaffinity-labeled Green Leaf Volatile Compound ‘Tricks’ Highly Selective and Sensitive Insect Olfactory Receptor Neurons
1 Laboratory of Chemical Prospecting, National Institute of Sericultural and Entomological Science, 1-2 Ohwashi, Tsukuba 305-8634, Japan and 2 Department of Entomology, University of California at Davis, Davis, CA 95616, USA
Correspondence to be sent to: Walter S. Leal, Department of Entomology, University of California at Davis, Davis, CA 95616, USA. e-mail: wsleal{at}ucdavis.edu
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Abstract |
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The sex pheromone of the scarab beetle, Phyllopertha diversa, is emitted by females and specifically detected by olfactory receptor neurons in the male and female antennae. Single sensillum recordings showed that, in contrast to the less sensitive pheromone sensilla in females, olfactory receptor neurons in the male antennae had a low threshold (1 ng), which rivals those of moths. The male and female antennae also possessed olfactory receptor neurons specific for the detection of floral and green leaf volatile compounds. Detectors for the green leaf volatile (Z)-3-hexenyl acetate had a threshold (10 pg) far below the sensitivity of the pheromone-detecting machinery. In addition, these neurons showed a remarkable selectivity even when challenged with related compounds at 10 000-fold higher concentrations. Surprisingly, a diazo analog, (Z)-3-hexenyl diazoacetate, elicited slightly higher nervous activity than the natural ligand in the neurons specific and selective for (Z)-3-hexenyl acetate. The inability of the green leaf volatile-detecting machinery to discriminate the photoaffinity-labeled compound from the natural product indicates that the synthetic ligand interacts with odorant-binding protein, odorant receptor and odorant-degrading enzyme as does the cognate ligand.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The selectivity and sensitivity of the olfactory system in insects 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 marked contrast to the olfactory receptor neurons specific for pheromones, insect detectors for chemicals others than pheromones (host plant volatiles and other semiochemicals) were earlier considered to be generalistic and relatively insensitive (Mason and Mustaparta, 1990), probably because the key stimuli have not been identified (Blight et al., 1995; Wibe and Mustaparta, 1996; Wibe et al., 1997, 1998; Hansson et al., 1999; Rostelien et al., 2000). Recently, we discovered in males of the pale brown chafer Phyllopertha diversa (Coleoptera: Scarabaeidae) olfactory receptor neurons specific for the detection of (Z)-3-hexenyl acetate and other green volatile compounds which displayed higher sensitivity and specificity than the pheromone detectors (Hansson et al., 1999).
In order to gain a better insight into the mechanisms underlying the binding and release of ligands in early olfactory processing, we have prepared photoaffinity-labeled ligands (Ganjian et al., 1978). Here, we report that, despite their remarkable selectivity and sensitivity, the detectors for (Z)-3-hexenyl acetate in the antennae of male and female P. diversa exhibit slightly higher sensitivity to an analog diazo compound.
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Materials and methods |
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Insects
Eggs laid by field-collected females of P. diversa were transferred to wet sand in ice cream cups. After hatching, the grubs were transferred individually to ice cream cups filled with moist leaf mould (Leal et al., 1994). Emerged adults were kept at 20°C and under a photoperiod of 16 h light and 8 h dark.
Single sensillum recordings
Beetles were immobilized in Eppendorf tubes cut at the bottom with the head sticking out of the hole and the antennae fixed with dental wax. The three lamellae of the antennae were separated and hold immobile with a thin metal pin. The indifferent (ground) electrode was a thin silver wire inserted in the abdomen. Extracellular contacts with antennal olfactory receptor neurons were established by inserting, under a stereomicroscope with up to 300x magnification, a tungsten electrode (sharpened electrolytically in KNO2 solution) through the cuticle adjacent to a sensillum placodeum (Hubel, 1957). The antennal preparation was continuously flushed with a charcoal-filtered and moistened air stream flowing at 0.5 m/s through an 8 mm i.d. glass tube ending 10 mm before the preparation. Each test compound was applied to a piece of filter paper (0.5 x 1 cm), which was dried for at least 10 min and inserted into a Pasteur pipette. Stimulation was performed by inserting the tip of the test pipette into a hole 6 cm from the outlet of the glass tube and air was blown for 300 ms through the pipette by a stimulus controller (CD-02/E; Syntech, Hilversum, The Netherlands). The signal was amplified using a Nihon Kohden MEZ-8300 amplifier (Tokyo, Japan). Antennal responses were stored using Axotape 2.0.2 (Axon Instruments, Foster City, CA) and visualized on a Nihon Kohden memory oscilloscope VC-11 (Tokyo, Japan). Spikes were counted manually from the computer screen. We used one-tailed interrupted time series analysis (Zacks, 1971; Hudson, 1977) to compare the number of action potentials occurring during consecutive 200 ms time intervals during a 5 s pre-stimulus (for an analysis of spontaneous activity) and during and after the initial 1 s of odorant stimulation of a single stimulus trial. The neural activity prior to exposure (pre-stimulation period) was compared with that during stimulation and the t value obtained was compared to the tabled critical t value (37%, P < 0.05). The results obtained were statistically processed (Zacks, 1971) and represented as diagrams.
Synthesis
(p-Toluenesulfonyl)hydrazonoacetic acid
To a solution of 90% glyoxylic acid (5 g, 0.049 mmol) in water (55 ml) at 55°C, p-toluenesulfonylhydrazide (11.6 g, 0.062 mmol) in hydrochloric acid (31 ml, 2.5 M) was added in small portions. The reaction mixture was stirred at 55°C for 1 h, then cooled to room temperature and kept in a refrigerator overnight. The crude p-toluenesulfonylhydrazone was collected on a filter, washed with cold water and air dried for 30 h. Tosyl hydrazone was recrystallized from carbon tetrachloride/ethyl acetate (2:1, 120 ml) and the product was air dried for 6 h to give 10.5 g of the hydrazone as white crystals (m.p. 150–152°C; yield, 70%).
(p-Toluenesulfonyl)hydrazonoacetyl chloride
To a suspension of (p-toluenesulfonyl)hydrazonoacetic acid (1.5 g, 8.06 mmol) in benzene (10 ml) was added thionyl chloride (1.2 ml, 16 mmol) dropwise and the reaction mixture was heated at reflux for 90 min with stirring. The reaction mixture was then cooled immediately, Celite (0.5 g) was added and the mixture was stirred for 3 min, filtered and concentrated under vacuum. The residue was triturated with warm benzene (1 ml), cooled to 5–10°C and filtered. The solid product was dissolved in warm benzene (1 ml), petroleum ether (3 ml) was added and the mixture was left overnight at room temperature. The precipitated crystals were collected by filtration and dried for 6 h to give 1.1 g of the desired acid chloride as pale yellow prisms (m.p. 103–108°C). The IR spectrum of the product showed peaks at 1775 cm–1 (shoulder) and 1740 cm–1 (C=O).
(Z)-3-Hexenyl diazoacetate
A solution of (p-toluenesulfonyl)hydrazonoacetyl chloride (0.51 g, 1.96 mmol) in methylene chloride (5 ml) was cooled in an ice bath. (Z)-3-Hexen-1-ol (0.190 g, 1.89 mmol; Wako, Japan) in methylene chloride (2 ml) was added to this cold solution and then a solution of triethylamine (0.28 ml) in methylene chloride (2 ml) was added to the cold reaction mixture dropwise with stirring. The resulting mixture was stirred at 0°C for 1 h, then a solution of triethylamine (0.32 ml) in methylene chloride (2 ml) was added to it. After stirring for 1 h at room temperature, the solvent was removed under reduced pressure with a rotary evaporator. The crude product (0.16 g, 50%) was purified by column chromatography (Florisil, benzene-ether 99:1). Pure (Z)-3-hexenyl diazoacetate was obtained by further purification on a silicic acid column eluted with hexane/ether (98:2). MS: m/z (relative intensity) 140 (1), 125 (2), 112 (3), 99 (10), 82 (65), 69 (93); 67 (100). Vapor phase FTIR: 3020 (sp2 C-H st), 2113 (C=N=N), 1730 (C=O), 735 cm–1 (cis C=C). 1H-NMR (CDCl3): 
0.9 (t, CH3), 1.97 (m, CH2-CH3), 2.3 (q, OCH2CH2), 4.07 (t, OCH2CH2), 4.66 (s, COCHN2), 5.2–5.8 (m, 2 vinylic protons). 13C NMR (CDCl3): 13.16, 19.58, 25.93, 28.66, 45.09, 63.33, 122.49, 131.71, 176.2.
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Results |
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Sex pheromone-specific detectors in male and female
We took recordings from a total of 108 sensilla in the antennae of male and female P. diversa, which utilizes a unique alkaloid pheromone, 1,3-dimethyl-2,4-(1H,3H)-quinazolinedione (Leal et al., 1997). Of 97 sensilla investigated in the female antennae, we found 21 olfactory neurons tuned to the pheromone. The dose–response trials with male and female antennae showed similar response profiles, but the female detectors were 100 times less sensitive (Figure 1). The olfactory receptor neurons tuned to the alkaloid pheromone in male and female P. diversa antennae did not respond to the green leaf volatile and floral compounds known as scarab attractants (Leal, 1998) and the sex pheromone of other scarab species, namely, (R)- and (S)-japonilure, (R)- and (S)-buibuilactone, methyl (Z)-5-tetradecenoate, (Z)-7-tetradecen-2-one, and (R,Z)-7,15-hexadecadien-4-olide (Leal, 1998). No response was observed even at concentrations up to 10 000-fold higher than the threshold for the alkaloid pheromone.
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Detectors for floral compounds
Thirty-three sensilla out of 97 investigated in the female antennae exhibited specificity for floral compounds. Although the threshold of the anethol-detecting olfactory receptor neurons (17 sensilla) was high (100 ng), they responded in a dose-dependent manner. In addition, they responded specifically to anethol. No response was observed when these detectors were challenged with high amounts (10–100 µg) of other floral, green volatile or pheromone compounds. Receptor neurons in two sensilla responded specifically to geraniol with performance similar to that of anethol detectors. On the other hand, 14 sensilla responded to phenethyl propionate with the lowest threshold observed for floral compounds (10 ng). However, when challenged with higher concentrations of the other test compounds, the phenethyl propionate detectors responded to geraniol in a dose-dependent manner (Figure 2).
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Green volatile detectors
Twenty-two sensilla in the female antennae responded to (Z)-3-hexenyl acetate in a dose-dependent manner and exhibited a very low threshold (Figure 3). These detectors were also stimulated by high concentrations of anethol. In contrast to the olfactory receptor neurons previously identified in males, which responded to (Z)-3-hexenol and (E)-2-hexenol at higher concentrations (Hansson et al., 1999), the female counterparts were completely silent to the related alcohols.
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Response to the photoaffinity-labeled green leaf volatile
(Z)-3-Hexenyl diazoacetate was prepared by reacting (Z)-3-hexen-1-ol with (p-toluenesulfonyl)hydrazonoacetyl chloride (Blankey, 1973) in the presence of triethylamine. The final compound was carefully purified by column chromatography and its purity (>99%) was checked by gas chromatography, high performance liquid chromatography and NMR. This compound stimulated the olfactory receptor neurons tuned to (Z)-3-hexenyl acetate in a manner similarly to the native ligand (Figure 4). Responses were tonic at low stimulus concentrations, phase-tonic at higher and gave a similar spike amplitude to that obtained with (Z)-3-hexenyl acetate. Comparison of the dose–response characteristics for (Z)-3-hexenyl acetate and its diazo analog using the same type of sensilla showed a slightly higher sensitivity to the photoaffinity-labeled analog (Figure 5). A similar dose–response curve was obtained with male antennae. On the other hand, (Z)-3-hexenyl diazoacetate did not stimulate olfactory receptor neurons of any other sensilla type, including the olfactory receptor neurons tuned to the sex pheromone, (Z)-3-hexenol (data not shown), and floral compounds.
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Discussion |
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The antennae of male and female P. diversa possess olfactory receptor neurons tuned to the sex pheromone released by the female. This scenario is found in many scarab species (Larsson et al., 1999
; Nikonov and Leal, unpublished data). In this study we found that the sensitivity of the female detectors in P. diversa was much lower (100-fold) than that of the pheromone-specific olfactory receptor neurons in male antennae. In addition, the number of sensilla placodea housing neurons specific for the pheromone is significantly lower in female than in male antennae. A lower number of pheromone-detecting sensilla on female than on male antennae has also been found in other phytophagous scarab species (Kim and Leal, unpublished data). The ability of females of some scarab species to detect their own sex pheromone is consistent with observations that the semiochemicals elicit behavioral responses in females (Leal et al., 1994, 1996). In contrast, we have never been able to observe, in the field or in indoor bioassays, any behavioral responses of female P. diversa to its own sex pheromone. Thus, the ability of the female to detect the alkaloid pheromone remains enigmatic.
In general, insect antennae can detect species-specific pheromones with high sensitivity and specificity. Recently, olfactory receptor neurons that are selective and sensitive to plant volatile compounds have also been characterized by gas chromatography linked to electrophysiological recordings from single receptor cells (Blight et al., 1995; Wibe and Mustaparta, 1996; Wibe et al., 1997, 1998; Hansson et al., 1999; Rostelien et al., 2000). Nevertheless, it is somewhat surprising that male and female P. diversa possess detectors highly tuned to ubiquitous green leaf volatile compounds which are far more sensitive than the pheromone-detecting machinery. Given this sensitivity and selectivity, the green leaf volatile-detecting machinery emerges as a model to study the molecular mechanisms underlying this impressive detector. The discovery of a diazo analog which mimics (Z)-3-hexenyl acetate so perfectly that the whole detecting machinery is tricked is fortuitous and opens new avenues for the understanding of insect olfaction. Photoaffinity-labeled insect pheromones have been widely used (Du et al., 1994) to study olfaction because they have been demonstrated to stimulate pheromone receptors (Ganjian et al., 1978). In contrast to the photoaffinity-labeled sex pheromone analog, which retained only 10% of the pheromone activity (Ganjian et al., 1978), the nervous activity elicited by (Z)-3-hexenyl diazoacetate was indistinguishable from that of the native ligand.
The transduction of chemical signals into neuronal activity in insect antennae is triggered by the interaction of cognate ligands with odor receptor proteins located in the dendritic membrane of olfactory receptor neurons (Clyne et al., 1999; Vosshall et al., 1999). However, prior to interaction with odor receptors, the airborne odor molecules must first enter the aqueous lumen of an olfactory sensillum. These hydrophobic ligands must then enter and pass through an aqueous environment containing very aggressive odorant-degrading enzymes (Vogt and Riddiford, 1981; Vogt et al., 1985), before arriving at the odor receptor molecules. This passage is accomplished through interaction between the odor molecules and odorant-binding proteins (OBPs). These proteins are thought to bind odor molecules at the interface between the aqueous lumen and the cuticular hair wall of the sensillum and to transport the odors by diffusion through the aqueous lumen to the neuronal membrane receptors.
Considering that (Z)-3-hexenyl diazoacetate elicits the same receptor response as the native ligand and, consequently, ‘tricks’ the whole detecting machinery, it may serve as an agonist to study interaction with OBPs, odorant receptors and/or odorant-degrading enzymes in the various steps of signal processing. Given that carbenes photolytically generated from diazoacetates (Ganjian et al., 1978) bind covalently to proteins (Du et al., 1994), photoaffinity-labeled semiochemicals may serve as probes to unveil details of perireceptor events, interaction with receptor proteins and inactivation of chemical signals.
Recently we identified two OBPs from the antennae of male and female P. diversa (Wojtasek et al., 1999). Although we believe that one of these OBPs binds green leaf volatile compounds, we were unable to demonstrate any binding activity, probably because the ligand evaporates during native gel bioassay. Attempts by other workers to detect binding of green leaf volatiles to lepidopteran proteins have also failed (Ziegelberger, 1996). Therefore, there is no direct evidence in the literature for the binding of semiochemicals to the so-called general OBPs (GOBPs). The only indirect evidence has been achieved by interaction of photoaffinity-labeled pheromone analogs with a recombinant GOBP (Feng and Prestwich, 1997). We hope that (Z)-3-hexenyl diazoacetate will allow clarification of the function(s) of the GOBPs.
Recent studies suggest that OBPs have dynamic structures, altering their conformations in pH-dependent ways (Wojtasek and Leal, 1999; Damberger et al., 2000; Leal, 2000; Sandler et al., 2000). These findings suggest a facilitated binding of odor molecules near the relatively neutral pH of the lumen–cuticle interface and facilitated unloading of odor molecules near the relatively acidic lumen– membrane interface in the proximity of the receptor proteins. However, the mechanism of this dynamic character of the OBPs is unknown. The covalent association of ligands with binding proteins may unravel further details of the molecular mechanism underlying the inordinate sensitivity and selectivity of the insect olfactory system.
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Acknowledgments |
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The Japanese component of this research was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (BRAIN) granted to W.S.L. A.A.N and J.T.V were supported by the BRAIN fund. We appreciate the helpful discussions of the members of our group.
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Accepted July 31, 2000
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