Responses of insect olfactory receptor neurons (ORNs) involve an entry of Ca2+ through olfactory heterodimeric receptor complexes. In moths, the termination of ORN responses was found to strongly depend on the external Ca2+ concentration through the activation of unknown Ca2+-dependent Cl− channels. We thus investigated the molecular identity of these Cl− channels. There is compelling evidence that bestrophins form Cl− channels when expressed in heterologous systems. Here we provide evidence that antennae of the moth Spodoptera littoralis express three transcripts encoding proteins with hallmarks of bestrophins. One of these transcripts, SlitBest1b, is expressed in ORNs. The heterologous expression of SlitBest1b protein in CHO-K1 cells yielded a Ca2+-activated Cl− current that shares electrophysiological properties with the native Ca2+-activated Cl− current of ORNs. Both currents are anionic, present similar dependence on the intracellular Ca2+ concentration, partly inactivate over time, have the same anion permeability sequence, the same sequence of inhibitory efficiency of blockers, the same almost linear I–V relationships and finally both currents do not depend on the cell volume. Therefore, our data suggest that SlitBest1b is a good candidate for being a molecular component of the olfactory Ca2+-activated Cl− channel and is likely to constitute part of the insect olfactory transduction pathway. A different function (e.g. regulation of other proteins, maintenance of the anionic homeostasis in the sensillar lymph) and a different role (e.g. involvement in the olfactory system development) cannot be excluded however.

Background

Insects respond to the spatial and temporal dynamics of a pheromone plume, which implies not only a strong response to 'odor on', but also to 'odor off'. This requires mechanisms geared toward a fast signal termination. Several mechanisms may contribute to signal termination, among which odorant-degrading enzymes. These enzymes putatively play a role in signal dynamics by a rapid inactivation of odorants in the vicinity of the sensory receptors, although direct in vivo experimental evidences are lacking. Here we verified the role of an extracellular carboxylesterase, esterase-6 (Est-6), in the sensory physiological and behavioral dynamics of Drosophila melanogaster response to its pheromone, cis-vaccenyl acetate (cVA). Est-6 was previously linked to post-mating effects in the reproductive system of females. As Est-6 is also known to hydrolyze cVA in vitro and is expressed in the main olfactory organ, the antenna, we tested here its role in olfaction as a putative odorant-degrading enzyme.

Results

We first confirm that Est-6 is highly expressed in olfactory sensilla, including cVA-sensitive sensilla, and we show that expression is likely associated with non-neuronal cells. Our electrophysiological approaches show that the dynamics of olfactory receptor neuron (ORN) responses is strongly influenced by Est-6, as in Est-6° null mutants (lacking the Est-6 gene) cVA-sensitive ORN showed increased firing rate and prolonged activity in response to cVA. Est-6° mutant males had a lower threshold of behavioral response to cVA, as revealed by the analysis of two cVA-induced behaviors. In particular, mutant males exhibited a strong decrease of male-male courtship, in association with a delay in courtship initiation.

Conclusions

Our study presents evidence that Est-6 plays a role in the physiological and behavioral dynamics of sex pheromone response in Drosophila males and supports a role of Est-6 as an odorant-degrading enzyme (ODE) in male antennae. Our results also expand the role of Est-6 in Drosophila biology, from reproduction to olfaction, and highlight the role of ODEs in insect olfaction.