This study demonstrates that a carboxylesterase, Est-6, previously linked to post-mating effects in the reproductive system of D. melanogaster females, plays also a role in the sensitivity and dynamics of ORNs tuned to cVA, the volatile fruit fly pheromone. We also infer that this physiological function of Est-6 at the peripheral olfactory level is required for normal male behavioral responses to cVA.
Est-6 is known as an extracellular enzyme in the male genital tract  and its extracellular location within the antennae has been shown by its isolation during the analysis of the soluble proteome of D. melanogaster antennae . We show here that Est-6 expression in male antennae is high and associated with most of olfactory sensilla, confirming the Est-6/lacZ pattern previously observed within the third antennal segment . In addition, we found that Est-6 expression is mostly associated with the accessory cells embedding the sensory neurons and located at the base of the sensilla. These cells are already known to produce OBPs and secrete them into the sensillum lymph [35–37]. Altogether, these data suggest that Est-6 could be secreted within the lymph of the olfactory sensilla, including cVA-sensitive sensilla.
As all other β-esterases, Est-6 clustered within a clade that includes extracellular catalytically competent esterases (reviewed in ). Phylogenetic analyses [13, 39] also revealed that Est-6 was closely related to the antennal carboxylesterases characterized in vitro in the wild silk moth Antheraea polyphemus [9, 12] and in the beetle Popilia japonica . These ODEs were able to hydrolyze the female sex pheromones in vitro with kinetics suggesting that they could play a significant role in the dynamic of signal termination in vivo [8, 9, 12]. cVA degradation by purified Est-6 into cis-vaccenyl alcohol (cVOH) has been shown in vitro  and it has been also demonstrated that cVOH elicits only very low responses in T1 sensory neurons . Together with its sensillar location, this catalytic activity towards the pheromone suggested that Est-6 could a play a role in pheromone signal termination, as a candidate ODE. Further determination of Est-6 kinetics towards cVA will be useful to precise its mode of action.
If ODEs were required for odorant processing, then their inhibition should disturb odorant reception within the antennae. Several pharmacological approaches have been used to address this question. Volatile trifluoroketones (TFKs), which can inhibit carboxylesterase activities  were used in several lepidopteran species to test their effect on pheromone response , but controversial effects were observed. In the moth Ostrinia nubilalis, prolonged repolarization time of EAG in response to the pheromone after TFK application suggested that esterases were involved in pheromone deactivation , but it has also been suggested that TFKs may interact with Ors, OBPs or other members of the transduction cascade [42, 43]. Inhibition of antennal cytochrome P450 by metyrapone reduces pheromone responses in a scarab beetle, suggesting that these intracellular enzymes were required for maintaining olfactory sensitivity .
In Drosophila, genetic tools offer the opportunity to knockdown candidate genes specifically to verify their physiological role directly. This approach led us to demonstrate that the absence of Est-6 in males indeed modifies neuronal responses to the pheromone, with stronger and longer-lasting responses. Noteworthy, the kinetics of signal termination within Est-6° T1 sensilla is altered even at physiological low doses of pheromone and with brief stimulations, as expected after the knockdown of an ODE. We can assume that the lack of Est-6 in mutant antennae prevents the degradation of cVA, which could lead to an accumulation of cVA in the perireceptor space of T1 sensilla. While binding with cVA, the OBP LUSH encounters a conformational change and the LUSH/cVA complex would be the active form that interacts with the receptor (Laughlin et al., 2008). LUSH increases the sensitivity of T1 ORN to cVA but does not to cVOH . In Est-6° mutant antennae, accumulation of cVA would thus lead to an accumulation of the complex OBP/pheromone, leading to stronger responses and delayed signal termination. However, Est-6 involvement in signal dynamics does not preclude a role of additional mechanisms in signal termination.
As Est-6 has a physiological effect on cVA reception, we thus tested whether Est-6 mutation could influence cVA-triggered behaviors. We found that Est-6 deficiency clearly enhances the antiaphrodisiac effect of the pheromone. Topical applications of exogenous cVA on females reduced male courtship as already observed . In addition, we showed that the effect of exogenous cVA is dose dependent in control and Est-6° males. However, the threshold of behavioral response to the pheromone is lower in Est-6° males. Compared to control flies, lower doses of cVA were sufficient to slow down courtship initiation of mutant males, thus to reduce their courtship. Est-6 deficiency also increases dispersal thus likely aggression. The proximity to a high density of male flies has been shown to increase the level of male aggression, thus dispersal, in a dose-dependent manner . Dispersal of Est-6° males in absence of exogenous cVA suggests again a lower threshold of behavioral response in mutant males.
Activation by cVA of ORNs carrying Or67d in T1 sensilla is sufficient to inhibit male-male courtship behavior , and to promote cVA-induced aggression . In particular, increasing artificially the excitability of Or67d-expressing ORNs, by expressing a bacterially-derived sodium channel, promotes dispersal of grouped flies even in absence of exogenous cVA . Modified physiological responses of T1 sensilla to cVA in Est-6° mutants could thus potentially account for the observed exacerbated behaviors. Delayed cVA termination and stronger responses of T1 sensilla from Est-6° mutant males are consistent with their lower threshold of behavioral response. Indirect evidence for a function specific for T1 sensilla comes from the fact that lack of Est-6 in mutants did not impair other chemically-driven behaviors, as indicated by a normal response to food odors and to female pheromones.