Pheromone reception in fruit flies expressing a moth's odorant receptor

Abstract

We have expressed a male-specific, pheromone-sensitive odorant receptor (OR), BmorOR1, from the silkworm moth Bombyx mori in an "empty neuron" housed in the ab3 sensilla of a Drosophila Deltahalo mutant. Single-sensillum recordings showed that the BmorOR1-expressing neurons in the transgenic flies responded to the B. mori pheromone bombykol, albeit with low sensitivity. These transgenic flies responded to lower doses of bombykol in an altered stimulation method with direct delivery of pheromone into the sensillum milieu. We also expressed a B. mori pheromone-binding protein, BmorPBP, in the BmorOR1-expressing ab3 sensilla. Despite the low levels of BmorPBP expression, flies carrying both BmorOR1 and BmorPBP showed significantly higher electrophysiological responses than BmorOR1 flies. Both types of BmorOR1-expressing flies responded to bombykol, and to a lesser extent to a second compound, bombykal, even without the addition of organic solvents to the recording electrode buffer. When the semiochemicals were delivered by the conventional puffing of stimulus on the antennae, the receptor responded to bombykol but not to bombykal. The onset of response was remarkably slow, and neural activity extended for an unusually long time (>1 min) after the end of stimulus delivery. We hypothesize that BmorOR1-expressing ab3 sensilla lack a pheromone-degrading enzyme to rapidly inactivate bombykol and terminate the signal. We also found an endogenous receptor in one of the sensillum types on Drosophila antenna that responds to bombykol and bombykal with sensitivity comparable to the pheromone-detecting sensilla on B. mori male antennae.

Fig. 1.

Action potentials (spikes) from ORNs within an ab4 sensillum on the antenna of Drosophila (Oregon R). Individual action potentials (A and B) denote spikes from two ORNs based on their amplitudes. Traces show responses of ab4A to solvent (a); increasing doses of bombykol from 0.01 (top trace), 0.1, 1, and 10 μg (bottom trace) (b); bombykal (10 μg) (c); and (E)-2-hexenal (10 ng) (d) by the puffing method. (Scale bar, 0.5-s stimulation.)

Fig. 2.

Expression of GFP in ab3 sensilla and transcripts of BmorOR1 and BmorPBP genes in the antennae of transgenic flies. (a) Proximomedial view of the antenna of an Or22a-Gal4xUAS-GFP fly under the fluorescent microscope, with GFP luminescence detected in the ab3 sensilla (Inset), which are brighter than the neighboring large basiconic sensilla ab1 and ab2. (Scale bar, 20 μm.) (b) Measurement of BmorOR1 gene expression in BmorOR1+BmorPBP flies by RT-PCR. Left, 10-antennae equivalent; Right, standard PCR products representing ≈10⁻²⁰, ≈10⁻²¹, ≈10⁻²², and ≈10⁻²³ mol of standard, in order from left to right. (c) BmorPBP gene expression in the same flies as in b. Left, 10-antennae equivalent; Right, standard PCR products representing ≈10⁻²⁰, ≈10⁻²¹, ≈10⁻²², and ≈10⁻²³ mol of standard, in order from left to right.

Fig. 3.

Action potentials recorded from the ab3A neuron in transgenic flies in response to solvent (Left) and to 10 μg of bombykol (Right). (a) Control flies. (b) Typical recordings from flies expressing only the OR from B. mori, BmorOR1. (c) Typical recordings from flies expressing BmorOR1 and BmorPBP in nearly equal molecular amounts. Note the irregular spontaneous spiking activity of the A cell (arrow). Representative traces from each BmorOR1-expressing genotype are shown to highlight variations in response intensity among sensilla and flies. (Scale bar, 2-s stimulus duration.) The irregular bursts (arrowhead) of the ab3A cells, as shown in solvent traces, are typical in flies with Δhalo background (13, 15).

Fig. 4.

Direct stimulation leads to increased response from ab3A. Action potentials from ab3A cells in response to (a) control containing 0.5% ethanol and (b) bombykol (≈38 ppm in 0.5% ethanol) in transgenic flies expressing BmorOR1. (c) Flies expressing both BmorOR1 and BmorPBP showed stronger response to the same dose of bombykol. We did not observe any changes in response magnitude up to many minutes during recording, thus suggesting that there was no shortage of stimulus supply. Each trace shows a 10-s recording starting immediately after contact was established.

Fig. 5.

Neural activity of ab3A cells in transgenic flies BmorOR1 and BmorOR1+BmorPBP in response to control containing 0.5% ethanol and to bombykol (≈38 ppm in 0.5% ethanol), recorded by the direct stimulation method. Treatments labeled with the same letter are not significantly different according to the Wilcoxon–Mann-Whitney rank-sum test. Spike counts were made for at least 100 s (length of each record) but are represented here for 1 s for consistency.

Fig. 6.

Differences in response kinetics as observed in simultaneous recordings of action potentials (upper trace; highpass filter) and receptor potentials (lower trace; lowpass filter). (a) Bombykol (10 μg) elicited a slow rising response from ab3A that lasted beyond the stimulus duration. (b) Heptan-2-one (1 ng) elicited a response in ab3B cells that rose and fell quickly with stimulation. (c) Response of ab4A cell stimulated with bombykol (10 ng). Note that the kinetics of the innate neuron (ab4A) responding to bombykol (c) was comparable to that of the ab3B cell activated by heptan-2-one (b) but differed remarkably from that of the Δab3A:BmorOR1 neuron (a). (Vertical scale bar, 16 mV for all receptor potentials.)

Fig. 7.

Responses of 10 different ab3A neurons to bombykol (10 μg) in each type of specified transgenic fly. Dots in each row depict individual spikes, whereas multiples rows show responses from different ORNs. Average firing rates in 500-ms bins for each genotype are shown in peristimulus time histograms. Note that the responses peaked toward the end of the 2-s stimulus period and remained higher than the levels of spontaneous firing activity at the prestimulus.