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Insect Sex-Pheromone Signals Mediated by Specific Combinations of Olfactory Receptors

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Science  11 Mar 2005:
Vol. 307, Issue 5715, pp. 1638-1642
DOI: 10.1126/science.1106267

Abstract

We describe two male-specific olfactory receptors (ORs) in the silk moth, Bombyx mori, that are mutually exclusively expressed in a pair of adjacent pheromone-sensitive neurons of male antennae: One is specifically tuned to bombykol, the sex pheromone, and the other to bombykal, its oxidized form. Both pheromone ORs are coexpressed with an OR from the highly conserved insect OR subfamily. This coexpression promotes the functional expression of pheromone receptors and confers ligand-stimulated nonselective cation channel activity. The same effects were also observed for general ORs. Both odorant and pheromone signaling pathways are mediated by means of a common mechanism in insects.

There are two distinct chemical perception mechanisms in the antennae of the insect olfactory system (1). The first perception mechanism, the “generalist” system, recognizes odorants from foods and plants. The olfactory receptor (OR) family, which belongs to the seven-transmembrane G protein–coupled receptor family and includes about 60 multigenes in insects, is responsible for the first step of chemosensation in the olfactory neurons of antennae (24). Studies of olfaction in fruit flies have shown that odorants are discriminated by a combinatorial receptor code mediated by broadly tuned ORs (5).

The other chemical perception mechanism is the “specialist” system, which detects pheromones that are elicited by conspecifics (6). Sex pheromones released by adult female insects are detected by narrowly tuned olfactory neurons in the conspecific male antennae. Notably, insects can discriminate a subtle difference in stereochemistry or chirality of molecules, and they also use the specific ratios of two or a few components as a species-specific cue (7). The silk moth, B. mori, possesses the simplest pheromone communication system, wherein one achiral compound, bombykol, elicits the full array of sexual behaviors (8).

We have recently cloned a male-specific OR gene, BmOR1, that encodes a sex-pheromone receptor in B. mori (9). Ectopic expression of BmOR1 in female antennae conferred electrophysiological responses to bombykol, providing evidence that BmOR1 is a bombykol receptor (9). The specific response to bombykol was also seen in Xenopus oocytes expressing BmOR1 and the G protein BmGαq. However, only 10 to 15% of the injected oocytes were responsive. Furthermore, high concentrations of bombykol were necessary to activate BmOR1. These observations led us to speculate that additional components are necessary to fully reconstitute the pheromone signaling pathway in a heterologous cell system.

Members of a highly conserved OR gene subfamily are expressed in many of the receptor neurons in the antennae of insects (4, 10, 11). OR neurons in Or83b mutant flies showed no odorant-evoked action potential and little spontaneous activity (12). Thus, the Or83b family does not seem to play a role in direct detection of odorants or pheromones but rather acts as a dimerization partner for pheromone ORs. BmOR2 in B. mori appears to be a member of this family of conserved ORs (9, 13). We thus examined the expression patterns of BmOR1 and BmOR2 in the male adult antenna by two-color double in situ hybridization. BmOR2 was broadly expressed in antennal neurons, and 43% of BmOR2-labeled cells coexpressed BmOR1 (Fig. 1A). Furthermore, all of the BmOR1-positive neurons expressed BmOR2, suggesting that these two ORs act together in sex pheromone detection in vivo. In addition, the level of BmOR1 was consistently higher in the membrane fraction of Xenopus oocytes coexpressing BmOR2 (fig. S1). Other members of the highly conserved family, including HR2, also enhanced the level of BmOR1 in the membrane fraction, whereas conventional ORs, such as HR6 (4), had no effect. These results suggest that BmOR2 ensures functional expression of BmOR1 by acting as a chaperone or an accessory protein.

Fig. 1.

Coexpression of BmOR1 and BmOR2 and electronic responses to bombykol. (A) Two-color fluorescent in situ hybridizations of 15-μm sections from a male antenna. BmOR1 [Digoxigenin-RNA (DIG-RNA)] is shown in green and BmOR2 (fluorescein RNA) in magenta. Scale bar, 20 μm. Out of 369 neurons expressing BmOR2 in 21 sensillum sections from five antennae, 159 neurons expressed BmOR1. (B) Bombykol-induced currents in Xenopus oocytes. (Top) Current traces of oocytes injected with the indicated cRNAs in response to 30 μM bombykol with depolarization step pulses. (Bottom) Current traces recorded at –80 mV. Lysophosphatidic acid (LPA) (1 μM) was used as a control for the induction of Ca2+-dependent Cl current in oocytes. (C) (Top) Current trace with application of the indicated concentrations of bombykol. Bombykol was sequentially applied to the same oocytes. (Bottom) The corresponding dose-response curve. Each point represents the mean current value (±SEM) from four independent oocytes. (D) Normal bath solution showed a reversal potential of –7.0 ± 2.0 mV (n = 5). For ion exchange experiments, currents obtained before bombykol application were digitally subtracted from the curves. Na+-free solution caused a negative shift of the reversal potential of –54.1 ± 1.1 mV (n = 4). High-K+ solution did not cause a significant shift in the reversal potential [–8.9 ± 1.9 mV (n = 5)]. High-Ba2+ solution resulted in a slight shift in the reversal potential to –50.1 ± 1.3 mV (n = 5). Error bars show mean ±SEM. (E) Effects of channel blockers on bombykol-induced currents. (Top) The current trace was recorded by applying bath solution containing 2-APB (50 μM) or RR (50 μM). (Bottom) Quantitative analysis of the inhibition of bombykol-induced currents by channel blockers. Data represent the means (±SEM) from five independent oocytes. [(B), (C), and (E)] Ligands were applied for 10 s at the time indicated by the arrowhead.

We next conducted two-electrode voltage clamp recording in Xenopus oocytes coinjected with complementary RNAs (cRNAs) encoding BmOR1, BmOR2, and BmGαq. Coexpression of BmOR2 increased the percentage of bombykol-responsive oocytes to more than 95% and induced larger currents than those in oocytes lacking BmOR2 (Fig. 1B). In the absence of BmOR2, the response amplitude (±SEM) at a holding potential of –80 mV and with 30 μM bombykol was 22.8 ± 3.2 nA (n = 6), whereas in the presence of BmOR2 and with 30 μM bombykol, the response amplitude was 2.1 ± 0.26 μA (n = 8) (Fig. 1B). Surprisingly, oocytes expressing BmOR1 and BmOR2 without BmGαq also showed a response to bombykol (Fig. 1B). This outward current with depolarizing pulses, however, was different from the slow activating Cl current that is due to BmGαq-mediated Ca2+ increases. Bombykol-induced increases in currents were dose dependent, with a 50% effective concentration EC50 of 1.5 μM. This is one order of magnitude smaller than that previously obtained in oocytes lacking BmOR2 coexpression (9) (Fig. 1C). Finally, the threshold concentration was approximately 100 nM. BmOR2 thus greatly enhances the sensitivity of BmOR1 to bombykol and initiates a previously unrecognized signaling cascade.

We next characterized the electrophysiological properties of bombykol responses in oocytes expressing BmOR1 and BmOR2 by current-voltage analysis. The current-voltage relationship of the bombykol-activated conductance was nearly linear with a reversal potential of –7.0 ± 2.0 mV (n = 5) (Fig. 1D). This channel property was different from that of the Ca2+-activated Cl channel in oocytes and thus indicated that a nonselective cation channel was involved in the response to bombykol. Current-voltage relationships with various extracellular ion compositions showed that the bombykol-induced current was carried preferentially by monovalent cations (Fig. 1D). The current induced by bombykol was substantially inhibited by ruthenium red (RR), a blocker of the inostitol-1,4,5-triphosphate–dependent channel and some transient receptor potential (TRP) channels, but not by 2-aminoethoxydiphenylborane (2-APB), which is an inhibitor for other TRP channels (14) (Fig. 1E).

We next examined whether other members of the Or83b family behaved similarly. The same nonselective cation channel activity in response to bombykol was observed when BmOR1 was coexpressed with HR2 or Or83b (87 and 62% amino acid sequence identity with BmOR2, respectively) but not upon coexpression with a conventional OR such as HR6 (Fig. 2A). To further determine whether the Or83b family plays the same role in general odorant responses, we assayed the responses in oocytes coexpressing Or83b and Or47a, a Drosophila OR whose ligand has been identified by in vivo expression (5). Oocytes expressing Or47a alone or Or83b alone showed no response to any of the cognate ligands for Or47a (Fig. 2B and fig. S2), whereas oocytes expressing both Or47a and Or83b responded to pentyl acetate and weakly to 2-heptanone but not to methyl salicylate or ethyl acetate (Fig. 2B). These results demonstrate that Or47a is a general OR, the ligand specificity of which appeared to be consistent with that of Or47a-expressing neurons (5). Thus, the Or83b family plays a common functional role in both odorant and pheromone signaling pathways, conferring nonselective cation channel activity, by interacting with conventional ORs (15). Oocytes coexpressing BmOR2 and the mouse eugenol receptor (mOR-EG) or the β2-adrenergic receptor did not respond to eugenol or isoproterenol, respectively, suggesting that the function of the Or83b family is limited to the insect ORs (Fig. 2C).

Fig. 2.

Effects of coexpression of the Or83b family on the pheromone or odorant responses in oocytes. Representative inward current recorded from oocytes injected with indicated cRNAs at a holding potential of –80 mV. A pheromone or an odorant was applied for 10 s at the time indicated by the arrowhead: 30 μM bombykol (A); 1 mM methyl salicylate, ethyl acetate, 2-heptanone, and pentyl acetate (B); or 10 μM isoproterenol for β2-adrenergic receptor (β2-AR) and 300 μM eugenol for mOR-EG (C).

We identified an additional 29 putative ORs in B. mori from the Silkworm Genome database, and we found that two of them, BmOR3 and BmOR4, were expressed specifically in the male antenna and that two receptors, BmOR5 and BmOR6, were dominantly expressed in males (Fig. 3A). Bombykal, an oxidized form of bombykol, another compound that is released from the female pheromone gland, elicits responses only in male antennae and has an inhibitory effect on bombykol-induced wing vibration and movement of male moths (16). We therefore tested both bombykol and bombykal in addition to hexadecanol (C16-OH) as a negative control to characterize the responses of these male-specific or male-dominant ORs. BmOR1-BmOR2 combination conferred a response to bombykol [10.0 ± 2.27 μA at 30 μM (n = 4)], a very weak response to bombykal [0.23 ± 0.05 μA at 30 μM (n = 4)], and no response to C16-OH (Fig. 3B). The BmOR3-BmOR2 combination elicited a response to bombykal [6.8 ± 0.15 μAat30 μM(n = 4)], a very weak response to bombykol [0.14 ± 0.02 μA at 30 μM (n = 4)], and no response to C16-OH (Fig. 3B). This effect of bombykal was dose dependent with an EC50 value of 0.26 μM (Fig. 3C), and the threshold concentration for activation was approximately 30 nM. The channel properties of the BmOR1-BmOR2 and BmOR3-BmOR2 combinations were similar, suggesting that the same signal transduction mechanism was involved for both. Neither bombykol nor bombykal activated BmOR4, BmOR5, or BmOR6. Furthermore, 41 odorants that had previously been reported to elicit responses in B. mori antennae (17) activated neither BmOR1 nor BmOR3 (18), suggesting that these two ORs are specialist pheromone receptors that possess a high degree of specificity. Ligands for BmOR4, BmOR5, and BmOR6 remain to be identified.

Fig. 3.

Characterization of male-specific BmOR genes in B. mori and identification of a bombykal receptor. (A) (Top) Phylogenic tree of moth ORs. The phylogenic tree was generated from an alignment of the entire amino acid sequences of moth ORs with the MEGA-2 program (Molecular Evolutionary Genetics Analysis v2.1, www.megasoftware.net). HR, Heliothis virescens OR (24); AgOR, Anopheles gambiae OR. (Bottom) Sex-specific expression pattern of BmOR genes in an antenna as determined by reverse transcription polymerase chain reaction. (B) Ligand specificity of BmORs. The current trace was recorded at –80 mV with sequential application of 30 μM hexadecanol (C16-OH), bombykal, and bombykol to the same oocyte expressing the indicated set of cRNAs. The chemicals were applied for 10 s at the time indicated by the arrowhead. The insets show a higher magnification of the current trace. (C) Dose-dependent responses to bombykal in oocytes expressing BmOR3 and BmOR2. (Top) Current trace recorded at –80 mV by application of the indicated concentration of bombykal. Bombykal was sequentially applied for 10 s to the same oocytes at the time indicated by an arrowhead. (Bottom) Dose-response curve of oocytes injected with BmOR3 and BmOR2. Each point represents the mean current value (±SEM) from four independent oocytes.

In situ hybridization showed that BmOR3 was colocalized with BmOR2 in olfactory neurons in the antennae of male moths (Fig. 4A). Of the BmOR2-positive cells, 48% expressed BmOR3. BmOR1 and BmOR3 were not colocalized and were mutually exclusively expressed in two adjacent olfactory neurons (Fig. 4A). The expression of BmOR1 and BmOR3 was localized to the olfactory neurons of a trichodea sensillum. This was confirmed by double in situ hybridization with pheromone-binding protein (PBP), which is expressed in the supporting cells that surround pheromone-sensitive neurons in the male moth antenna (19) (Fig. 4B). Our results are consistent with previous physiological studies in which one of a pair of pheromone-sensitive neurons in a long sensillum trichodeum was activated by bombykol and the other responded to bombykal (16). PBP is involved in pheromone detection and possibly pheromone discrimination (20). Thus, PBP may play a role in dissolving pheromones in the sensillum lymph, thereby lowering the threshold concentration necessary for BmOR1 activation.

Fig. 4.

Expression pattern of BmOR1 and BmOR3 in an antenna of an adult male moth. (A) Two-color fluorescent in situ hybridization of 15-μm sections of an antenna. Scale bar, 20 μm. (Top) Localization of BmOR2 (fluorescein RNA, green) and BmOR3 (DIG-RNA, magenta). Out of 187 neurons expressing BmOR2 in eight sensillum sections from five antennae, 90 neurons expressed BmOR3. (Middle) Localization of BmOR1 (fluorescein RNA, green) and BmOR3 (DIG-RNA, magenta). (Bottom) Higher magnification images of the boxed regions in the middle panels. (B) Schematic drawing of olfactory neurons and supporting cells (SC) in a pheromone-sensitive sensillum (SL). The expression patterns of BmORs and PBP are indicated by the following colors: BmOR1, magenta; BmOR3, red; PBP, green. CU, cuticle. Two-color fluorescent in situ hybridization of PBP (fluorescein RNA, green) and either BmOR1 (DIG-RNA, magenta) or BmOR3 (DIG-RNA, red). Scale bar, 5 μm. (C) Model of OR-mediated signal transduction of odorant or pheromone responses in insects. Each neuron expresses one type of conventional OR that is coexpressed with a member of the Or83b family. Conventional ORs bind their cognate ligands (pheromone or odorant), resulting in stimulation of a G protein–mediated pathway and activation of a nonselective channel by coupling with the Or83b family. Transmembrane topology was predicted by the TMHMM program (TransMembrane Hidden Markow Model, www.cbs.dtu.dk/services/TMHMM).

Specialist neurons express a single type of male-specific OR that appears to possess a narrow specificity for pheromones: BmOR1 for bombykol and BmOR3 for bombykal. This mutually exclusive expression pattern of two pheromone receptors in single sensillum may provide a common paradigm to ensure detection of the specific ratios of a pheromone blend in the moth antenna. The neurons expressing pheromone receptors are projected into the macroglomerular complex in the male brain where pheromone information is further integrated to elicit the behavioral movement (21, 22). In contrast, generalist neurons express other ORs that detect general odorants (5), and the combinatorial code for each odorant is transmitted to distinct areas in the brain (23). Both specialist and generalist neurons require coexpression of the Or83b family, which appears to promiscuously couple with conventional ORs and ensure their chemoreceptor function by mediating nonselective cation channel activity. Although clarification of the in vivo mechanisms underlying OR-mediated current generation awaits further molecular and physiological analysis, this atypical function of a seven-transmembrane receptor suggests that there are unique and previously unappreciated aspects of receptor signal transduction.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1106267/DC1

Materials and Methods

Figs. S1 and S2

References

References and Notes

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