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A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in Drosophila

Thomas Chertemps, Adrien François, Gloria Rosell, Teun Dekker, Philippe

Lucas, Martine Maibeche Coisne, Nicolas Durand

► To cite this version:

Thomas Chertemps, Adrien François, Gloria Rosell, Teun Dekker, Philippe Lucas, et al.. A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in Drosophila. BMC Biology, 2012, 10, 10.1186/1741-7007-10-56. doi:10.1186/1741-7007-10-56

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# A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in *Drosophila*

Thomas Chertemps¹, Adrien François¹,², Nicolas Durand¹, Gloria Rosell³, Teun Dekker⁴, Philippe Lucas² and Martine Maïbèche-Coisne¹*

## Abstract

**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.

**Keywords:** carboxylesterase, esterase 6, olfaction, pheromone, signal termination

## Background

A sense that lacks spatial resolution requires a high temporal resolution for accurate location of signal sources in space. Insect pheromone responses exemplify this, with a capability of resolving and responding to pheromone filaments in 100 to 200 ms [1]. For such a system to operate it requires not only fast responses to

'odor on', but also to 'odor off'. Odor-off responses imply inactivation of odorant signals. Several mechanisms have been proposed to participate in signal cessation or reduction within insect olfactory hairs (or sensilla), involving either olfactory receptors (Ors) or molecules interacting with them (reviewed in [2]). In *Drosophila melanogaster*, when Or genes were expressed in another olfactory receptor neuron (ORN) than their native ORN by using the 'empty neuron' system (Δhalo mutant), signal termination was similar to what had been observed in their native ORN, suggesting that Ors

<footnote>* Correspondence: martine.maibeche@snv.jussieu.fr
¹Université Pierre et Marie Curie, UMR 1272, Physiologie de l'Insecte, Signalisation et Communication, F-75252, Paris, France
Full list of author information is available at the end of the article</footnote>
play a key role in signal dynamics [3]. However, when
an Or from the silk moth *Bombyx mori* was expressed
in *Drosophila* T1 sensilla, termination of the response
was rapid, whereas it was delayed when this receptor
was expressed in another type of sensilla than the T1s,
suggesting that the cellular environment of Ors could
also play a role in the dynamics of the response [4]. In
particular, fast degradation of odorants in the vicinity of
Ors by odorant-degrading enzymes (ODEs) has been
proposed as a mechanism contributing to the termina-
tion of ORN responses. Pheromone degradation *in vitro*
by antennal extracts ([5]; reviewed in [6,7]), by purified
antennal enzymes [8,9], as well as enzymatic inhibition
*in vivo* (reviewed in [10,11]) strongly support this
hypothesis. Various enzyme families were described as
candidate ODEs, such as carboxylesterases, aldehyde
oxidases, epoxide hydrolases, glutathione-S-transferases
or cytochrome P450 (reviewed in [7]). Few ODEs have
been both identified at the molecular level and function-
ally characterized *in vitro*. Among them, carboxyles-
terases involved in pheromone/odorant degradation
were the most studied [8,9,12-14]. However, involve-
ment of ODEs in odorant processing has never been
directly demonstrated *in vivo*.

In *Drosophila melanogaster*, the molecular mechanisms involved in the reception of a male-produced olfactory pheromone, *cis*-vaccenyl acetate (cVA), are intensely studied. In males, cVA suppresses male-male courtship [15] and promotes male-male aggression [16]. cVA also suppresses courtship towards recently-mated females, as cVA is transferred to the female with the seminal fluid [17]. Or67d, an Or mediating the sensory and behavioral responses to the cVA [15,16,18,19] is expressed in T1 trichoid sensilla [18]. LUSH, an odorant-binding protein (OBP), and SNMP1, a putative membrane bound coreceptor, are also required for cVA sensitivity [15,18,20-22]. Elements of binding and reception of cVA within T1 sensilla are thus well documented, but the mechanisms of cVA inactivation are unknown. In our search for putative factors that are involved in cVA degradation we noted that an extracellular carboxylesterase (EC 3.1.1.1), Esterase-6 (Est-6; CG6917), which is transferred during copulation to the female with the seminal fluid [23], hydrolyzes cVA *in vitro* [24]. Interestingly in males, Est-6 is not only expressed in the ejaculatory duct [25], but also in the antennae [26-29], suggesting that Est-6 could play a role in pheromone processing.

In the present work we determined *in vivo* the role of
*Est-6* in cVA olfaction. We studied the phenotypes of
several *Est-6* mutant and control strains at the electro-
physiological and behavioral levels. Our results demon-
strate that *Est-6* enables flies to detect and respond to
the temporal dynamics of cVA stimulation. In addition,

cVA-triggered behaviors are also modified in mutants,
suggesting that *Est-6* is of behavioral significance.

Results

Est-6 is highly and broadly expressed in male antennae

First we quantified the transcript levels between differ-
ent chemosensory appendages by quantitative PCR
(qPCR). *Est-6* levels were ninefold higher in antennae
compared to the proboscis (gustatory organ)/maxillary
palps (olfactory organ) (Figure 1A). *Est-6* was barely
detectable in legs, which bear gustatory sensilla, includ-
ing sensilla responding to female-specific pheromones
[30]. Interestingly, we also observed a clear sexual
dimorphism, as male antennae expressed 6.5-fold more
*Est-6* than the female antennae. To examine the expres-
sion pattern of *Est-6* within chemosensory organs, we
observed *Est-6-Gal4/UAS-mCD8-GFP* male antennae,
which express green fluorescent protein (GFP) under
the control of *Est-6* promoter. *GFP* was widely
expressed throughout the third antennal segment (Fig-
ure 1B, C). Moreover, most of the GFP+ cells did not
seem to coexpress ELAV, a neuronal marker (Figure 1D,
F). *Est-6* is thus highly and broadly expressed in male
antenna. At the cellular level, a neuronal expression
could not be completely excluded, but expression is
mostly observed in olfactory accessory cells surrounding
ORNs.

ORN responses to cVA depend on *Est-6* expression

To test whether the olfactory response to cVA is modi-
fied in *Est-6*° males, we first recorded the responses of
antennae by electroantennography (EAG). A dose
response curve to cVA was established (Additional file
1, Figure S1). We selected a dose of cVA (200 µg/car-
tridge) that induced a high response with our system
and performed long stimulation (5 s) in order to mimic
an overstimulation of the antennae. In these conditions,
the dynamics of EAG responses clearly differed between
the *null* mutant *Est-6*°, which completely lacks *Est-6*,
and the two control strains, that is, the wild-type strain
*Canton S* ('*CS*') and the rescue strain ('*Rescue*'), in which
*Est-6* expression was restored (Figure 2A): the depolari-
zation was similar in the three strains but the repolari-
zation was slower in *Est-6*° males. The repolarization
rates at the end of the stimulation (Figure 2B) were
reduced in *Est-6*° mutant compared to the controls (8%
in *Est-6*° vs 22.4% in *CS* and 20.7% in the rescue strain).
EAG results thus indicate that the lack of *Est-6* in
mutant flies affects the temporal dynamics of antennal
responses to cVA, with a delayed signal termination. To
test whether the *Est-6* mutation affects the general func-
tioning of the antennae, we measured their responses to
a different odorant. We selected 2-heptanone, because
this ketone is detected by a basiconic type sensillum
<figure><img src="image_3.png" /><figcaption>**Figure 1** *Est-6 expression in antennae.* (A) Quantitative PCR (qPCR) analysis of *Est-6* expression on cDNAs from wild-type (CS) male and female antennae, male gustatory appendages and heads without sensory appendages. *Est-6* expression level was normalized to that of *pgk*. Data were obtained from triplicate biological experiments and are given as the mean ± SEM. (B) Third antennal segment, posterior view of left antenna. Medial is to the left. (C) Expression of *Est-6* in *Est-6*<sup>Gal4</sup>/UAS-mCD8-GFP male. (D) Anti-ELAV staining of *Est-6*<sup>Gal4</sup>/UAS-mCD8-GFP antennae. (E) Merge of (C, D). (F) higher magnification of (E). Scale bars = 20 µM (except for (F), scale bar = 8 µm).</figcaption></figure>

that is distinct from cVA-responding trichoid sensilla
[31] and because its chemical structure prevents degra-
dation by esterases. In contrast to cVA, high doses of 2-
heptanone did not elicit different responses between
control and *Est-6*° males (Figure 2C, D), suggesting that
the *Est-6* mutation does not have a general effect on the
olfactory detection.

As the relationship between EAGs and response charac-
teristics of underlying sensory neuron is tenuous, we
therefore verified the temporal response characteristics
of individual ORNs in T1 sensilla, which are specifically
tuned to cVA, using single-sensillum recordings (SSR).
We used physiological doses of cVA and brief stimula-
tions as well as higher doses and long stimulations. Both
firing rate and response duration of T1 neurons upon
stimulations with 5 and 50 µg of cVA were affected in
*Est-6*° mutants compared to CS and *Rescue* flies (Figure
3A, B). With 0.5-s stimulation and both doses of cVA,
*Est-6*° mutants exhibited a delayed signal termination, as
revealed by the increased spiking rate after stimulation

(4.4 spikes/s in *Est-6*° vs 0.9 in *CS* and 1.0 in *Rescue* flies for a dose of 5 µg; 13.5 spikes/s in *Est-6*° vs 2.9 in *CS* and 4.7 in *Rescue* flies for a dose of 50 µg). With prolonged, 3-s stimulations, at low and high doses of cVA, both firing rate and duration of the response were significantly increased in mutant males compared to *CS* and *Rescue* strains (Figure 3B). In summary, following cVA stimulation, T1 sensilla in *Est-6*° males responded with a delayed signal termination, whatever the dose and the duration of the stimulation. In addition, firing rate during stimulation was also increased in response to prolonged stimulations.

*Est-6 modulates courtship behavior*

To examine whether *Est-6* mutation could influence
cVA-induced behaviors, we first measured male-male
sexual behavior by measuring the courtship index (CI)
toward a *CS* target male. Under daylight conditions and
when paired with an immobilized male, a wild-type
male courts typically with a CI of 10% [15]. This
<figure><img src="image_4.png" /><figcaption>**Figure 2** Antennal responses of males to cis-vaccenyl acetate (cVA) and heptanone. (A) Average electroantennography (EAG) plots from wild-type (CS, in black), null mutant (*Est*-6°, in red) and rescue (*Rescue*, in grey) strains during a 5-s stimulation with cVA. The horizontal bar indicates the duration of stimulus delivery. (B) Percentage of repolarization from the same genotypes calculated at the end of a 5-s stimulation with cVA. Mean ± SEM; *N* ≥ 10 for each genotype and test. (C) Average EAG plots from CS, *Est*-6° and *Rescue* male flies during a 5-s stimulation with heptanone. (D) Percentage of repolarization from the same genotypes calculated at the end of a 5-s stimulation with heptanone. Mean ± SEM; *N* = 13 for each test. Student's t test, \*P* < 0.05; \*\*P* < 0.01; \*\*\*P* < 0.001.</figcaption></figure>

percentage increased significantly when visual cues were suppressed (CI of 25 to 28% in control strains, Figure 4A). The CI was however strongly reduced in *Est*-6° (6.5%). Similarly, copulation attempts were absent in *Est*-6° mutants (Figure 4B), indicating a decrease in courtship vigor. Conversely, the latency of wing vibration was higher in the mutant strain (Figure 4C), indicating a delay in courtship initiation. In the rescue strain, all these phenotypes were restored. Absence of *Est*-6 thus correlated with a decreased male-male courtship, suggesting that the mutation enhances the antiaphrodisiac effect of cVA.

Courtship of males is thought to be inhibited by cVA [17,32], however, male gustatory pheromones such as Z-7-tricosene are also antiaphrodisiac for males [33]. To evaluate whether the behavioral modification observed in *Est*-6 mutant flies could be directly linked to cVA, we analyzed the heterosexual courtship of males. Under dim red light, the CI of CS and *Rescue* males to CS

decapitated virgin females ranged between 53.2% to 57.6%, respectively, and did not significantly differ from those of *Est*-6° mutant males (45.4%) (Figure 5A). Copulation attempts (Figure 5B) and courtship initiation (Figure 5C) was also comparable in the three strains. Absence of *Est*-6 had thus no effect on heterosexual courtship, suggesting that the perception of female pheromones is not affected. As heterosexual courtship is driven by gustatory and olfactory cues, this result suggested that *Est*-6 mutation did not interfere with their detection and integration.

We subsequently scored the CI of males to CS virgin females treated with exogenous cVA or solvent only. With solvent only, the CI was comparable in CS, *Rescue* and *Est*-6° males (53.4%, 50.8% and 52.8%, respectively; Figure 5A) and reached similar level as without any treatment. With the highest dose of cVA, the CI of control and *Est*-6° flies dropped to around 14%. The intermediate dose of 150 ng (approximately two-thirds male
<figure><img src="image_5.png" /><figcaption>**Figure 3** T1 sensillum responses to cis-vaccenyl acetate (cVA) measured by single-sensillum recordings. (A) Action potentials recorded from CS and *Est*-6° T1 sensilla stimulated for 3 s with solvent (left) or cVA (right). Horizontal bars indicate the duration of stimulus delivery. (B) Dose-response curves for T1 sensilla from CS, *Est*-6° and *Rescue* male flies: spike frequencies during the stimulation (0.5 s and 3 s), as well as for 9 s following the stimulus were calculated relatively to the prestimulus frequency. Mean ± SEM; N ≥ 8 for each data point. Student's t test, \*P < 0.05; \*\*P < 0.01.</figcaption></figure>

equivalent) induced a decrease of courtship in the three genotypes, however this decrease was more pronounced in *Est*-6 (19.2%) than in *CS* (39.8%) and *Rescue* (32,2%) males. This decrease was associated with a delay in courtship initiation, as shown by the corresponding wing vibration latency (Figure 5C). More interestingly, with the lowest dose (50 ng, 1/4 male equivalent) the CI of *CS* and *Rescue* males was unaffected, whereas it was

significantly reduced in *Est*-6° males (30.4%), with again a delay in courtship initiation. A decrease in copulation attempts was observed when females were treated with cVA, but this decrease was comparable in the three strains (Figure 5B). The antiaphrodisiac effect of cVA was thus dose-dependent in the three strains. However, the amount of synthetic cVA required to inhibit the male courtship was lower for *Est*-6° than for control
A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<figure><img src="image_8.png" /></figure>

**Figure 4 Analysis of male-male courtship.** The courtship index (CI) is the fraction of time spent in courtship activity in the 10-minute observation period. (A) CI of males of the indicated genotypes paired with a decapitated CS target male. (B) Number of copulation attempts. (C) Wing vibration latency in seconds. Mean ± SEM; N ≥ 30 for each genotype and test. Mann Whitney, *P* < 0.05; **P** < 0.001.

males, suggesting that the mutants presented a lowest threshold of response to the pheromone.

### Est-6 modulates aggression-promoting behavior

As cVA also promotes male-male aggression [16], we finally compared aggression behavior between Est-6° and control males. Male aggressiveness was analyzed indir-ectly, using a dispersal test. The dispersal of male flies competing for a food resource is indeed correlated with the level of aggression [16]. In the absence of synthetic cVA, control males quickly aggregated on the food resource and remained there for at least 30 minutes after introduction into the chamber (Figure 6). Solvent only (acetone) did not have any effect. In the presence of synthetic cVA at high dose (500 µg) after initial attraction to the resource, the number of CS and Rescue flies on the food cup declined, indicating aggression-induced dispersal. Est-6° males exhibited also increased dispersal, but this behavior was observed even in absence of synthetic cVA. As cVA is a volatile pheromone, its concentration is proportional to the number of male flies in a given environment. Our results suggest that the level of cVA emitted in the test chamber by six males was sufficient to trigger dispersal of Est-6° males, but not of control males.

### Olfactory behavior in response to food odors is not altered in mutant flies

The response of male flies to olfactory cues from food was evaluated using a trap assay [34]. Mutant flies were able to detect and locate the food paste equally well as control males (Additional file 2, Figure S2A). As the performance in this trap assay is a good reflect of the olfactory function [34], the result indicates that the lack

of Est-6 in the antennae or the genital tract does not have a general effect on olfactory-driven behaviors in mutants. Finally, the locomotor activity of mutant males was also comparable to control males, which demon-strates that Est-6 mutation does not affect locomotor activity in general (Additional file 2, Figure S2B).

### Discussion

This study demonstrates that a carboxylesterase, Est-6, previously linked to post-mating effects in the reproduc-tive 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 olfac-tory level is required for normal male behavioral responses to cVA.

Est-6 is known as an extracellular enzyme in the male genital tract [23] and its extracellular location within the antennae has been shown by its isolation during the analysis of the soluble proteome of *D. melanogaster* antennae [29]. We show here that Est-6 expression in male antennae is high and associated with most of olfac-tory sensilla, confirming the Est-6/lacZ pattern pre-viously observed within the third antennal segment [28]. In addition, we found that Est-6 expression is mostly associated with the accessory cells embedding the sen-sory 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]. Alto-gether, 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
<figure><img src="image_9.png" /><figcaption>**Figure 5** Analysis of male-female courtship. (A) CI of males of the indicated genotypes paired with a decapitated CS target virgin female. Females were treated either with acetone, 50, 150 or 400 ng of synthetic cis-vaccenyl acetate (cVA). (B) Number of copulation attempts. (C) Wing vibration latency in seconds. Mean ± SEM; N ≥ 30 for each genotype and test. Mann Whitney, *P* < 0.05.</figcaption></figure>

esterases (reviewed in [38]). 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* [8]. These ODEs were able
to hydrolyze the female sex pheromones *in vitro* with
<figure><img src="image_10.png" /><figcaption>**Figure 6** Dispersal test of male flies from a food resource. Number of flies of the indicated genotype on the food cup in the absence or presence of synthetic cis-vaccenyl acetate (cVA). Dispersion reflects the aggressiveness level under group-housing conditions. CS: dark dashed line; CS + acetone: dark dotted line; CS + cVA: dark bold line; Rescue: grey dashed line; Rescue + acetone: grey dotted line; Rescue + cVA: grey bold line; *Est-6°*: red dashed line; *Est-6°* + acetone: red dotted line; *Est-6°* + cVA: red bold line. N ≥ 8. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test.</figcaption></figure>

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* [24] and it has
been also demonstrated that cVOH elicits only very low
responses in T1 sensory neurons [40]. Together with its
sensillar location, this catalytic activity towards the pher-
omone suggested that Est-6 could a play a role in pher-
omone signal termination, as a candidate ODE. Further
determination of Est-6 kinetics towards cVA will be use-
ful 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 trifluoroke-
tones (TFKs), which can inhibit carboxylesterase activ-
ities [12] were used in several lepidopteran species to
test their effect on pheromone response [10], but con-
troversial effects were observed. In the moth *Ostrinia
nubilalis*, prolonged repolarization time of EAG in
response to the pheromone after TFK application sug-
gested that esterases were involved in pheromone deac-
tivation [41], 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 intra-
cellular enzymes were required for maintaining olfactory
sensitivity [11].

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 phero-
mone 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 [21]. In *Est-6°*
mutant antennae, accumulation of cVA would thus
lead to an accumulation of the complex OBP/phero-
mone, 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 phero-
mone. Topical applications of exogenous cVA on
females reduced male courtship as already observed
[15]. In addition, we showed that the effect of exogen-
ous 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 con-
trol 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-dep-
dent manner [16]. 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 beha-
vior [15], and to promote cVA-induced aggression [16].
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 [16]. 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 beha-
vioral 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 beha-
viors, as indicated by a normal response to food odors
and to female pheromones.

Conclusions

In conclusion, we have demonstrated that an extracellular esterase, Est-6, is involved in maintaining proper temporal dynamics of cVA detection at the peripheral olfactory circuit level and is involved in cVA-induced behaviors in males. These results expand the role of Est-6 in *Drosophila* biology, from reproduction to olfaction. After its transfer during mating with the semen, Est-6 is known to rapidly translocate to the female hemolymph and to impact female reproductive behavior (stimulation of egg laying and inhibition of receptivity for remating) [23]. Our results demonstrate that the same enzyme plays a crucial role in cVA detection in male antennae. This work also highlights the physiological role of carboxylesterases in insect odorant reception *in vivo*. In vertebrates, a potential role of extracellular enzymes from the nasal mucus has been recently revealed by a pharmacological inhibition approach [44]. Enzymatic conversion of odorants seemed to be fast enough to affect olfactory dynamics. The corresponding enzymes were not characterized, but carboxylesterases were suspected to play a role in ester conversion [44]. Enzyme-based mechanism of inactivation could be comparable in olfactory systems of insects and vertebrates.

In a context of pest insect management, these
enzymes may be interesting targets for the development
of specific inhibitors that interfere with the insect's abil-
ity to respond adequately to olfactory cues from mates
or host plants.

**Methods**

Fly strains, rearing and tissue collection

The following strains were used during this study: an
*Est-6*° null mutant strain (Bloomington stock 4211),

completely lacking *Est-6* and described in detail [23]; a
rescue strain described in Odgers *et al*. [45], which pre-
sents a similar genetic background as *Est-6°* (the *Est-6*
promoter was fused to the *Est-6* coding region and
transformed into the *Est-6 null* background); Canton-S
(CS) flies were used as wild-type control flies. Trans-
genic *UAS-mCD8::GFP* (Bloomington stock 5130) flies
were used for immunohistochemistry experiments to
determine more precisely the expression pattern of *Est-6*
within antennae. For the generation of the *Est-6 Gal4*
lines, a 1,132 fragment corresponding to the *Est-6* pro-
moter region was cloned in the pChs-Gal4 vector, and
transgenic flies were generated by P-mediated germline
transformation by BestGene Inc. (Chino Hill, CA, USA)
according to standard procedures [46]. All flies were
raised at 25°C on standard yeast/cornmeal/agar medium
in a 12-h light/12-h dark cycle, 50% to 60% relative
humidity.

qPCR

To precisely define the levels of *Est-6* expression in
antennae and other chemosensory appendages, *Est-6*
transcripts were quantified by qPCR analysis. Antennae
from 5 to 7-day old males and females, male legs and
proboscis with maxillary palps were dissected for total
RNA extraction using TRIzol Reagent (Invitrogen, Carls-
bad, CA, USA). Heads without appendages were also
tested. Single-stranded cDNAs were synthesized from
total RNAs (1 µg) using Superscript II reverse transcrip-
tase (Invitrogen). All reactions were performed as pre-
viously described [47] on the LightCycler® 480 Real-
Time PCR System (Roche, Basel, Switzerland). Each
reaction was run in triplicate with at least three inde-
pendent biological replicates. The *pgk*, *rpl8* and *rp49*
genes were used as reference genes. Specific primers
were designed using EPRIMER3 http://mobyle.pasteur.fr
and were as follows: *rp49up* CGGATCGATATGC-
TAAGCTGT, *rp49do* ACGTTGTGCACCAGGAACTT,
*rpL8up* TCGTATCGACAAGCCCATCCTGA, *rpl8do*
ACCACGGATCCTACCGGTACGAC, *pgkup* CGA-
GAAACTGGTGGAGAAGG, *pgkdo* CGAAGTTGGG-
GAACTCAAAG, *Est6up* TTCCCGGAAACTATG
GACTG and *Est6do* CAGTTCAAAGGCTCGTCCTC.
Normalized *Est-6* expression was calculated with Q-
Gene software [48].

Localization of *Est-6* expression within antennae

To localize the expression site of *Est-6* in the antenna,
we used transgenic flies expressing GFP under the con-
trol of *Est-6* promoter and we performed immunohisto-
chemistry with an anti-ELAV antibody as neuronal
marker. Heads with antennae from 5-day-old males *Est-
6 Gal4 /UAS-mCD8-GFP* were fixed for 3 h in 4% parafor-
maldehyde with 0.2% Triton X-100 (Sigma-Aldrich,
Saint-Quentin Fallavier, France), then washed for 1 h with phosphate-buffered saline containing 0.2% Triton X-100 (PBST, Sigma-Aldrich). Heads were then embedded in Tissue-Tek™ (CellPath, Newtown Powys, UK) and cryosections (15 µm) were set in cell culture insert (Greiner Bio-one, Monroe, LA, USA). After blocking with 3% normal goat serum and 1% bovine serum albumin (BSA) in PBST (1 h at room temperature (RT)), an anti-ELAV (from Developmental Studies Hybridoma Bank, University of Iowa) was diluted 1:10 (v/v) in the blocking solution (3% normal goat serum in PBST) and incubated overnight at RT. After a brief rinse in PBST, an anti-mouse conjugated Alexa-546 (Invitrogen) was applied 1:250 (v/v) in the blocking solution for 4 h at RT. tissues were mounted in Slow-fade reagent (Invitrogen). Imaging was performed on Olympus BX61 microscope with a ScopePro software.

## Comparison of antennal responses to cVA by EAG

EAG recordings were performed at RT on 5-day-old males previously kept in individual tubes, as described previously [49]. Reference and recording glass capillary electrodes were filled with 120 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 4 mM MgCl₂, 10 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES, Sigma-Aldrich), pH = 7.2. The reference electrode (approximately 1 µm tip diameter) was inserted in one eye and the recording electrode (approximately 10 µm tip diameter) was pushed against one antenna. The signal was amplified (×500) and low pass filtered online (10 kHz) with an Axopatch 200B amplifier (Molecular Devices, Union City, CA, USA) and digitized at 1 kHz with a Digidata 1440A acquisition board (Molecular Devices). A dose-response curve was established to select a dose of cVA that induces a high and saturating response. Antennae were then stimulated for 5 s with either pure hexane (> 98% purity, Carlo-Erba Reagents, Val de Reuil, France) as negative control, or with cVA (diluted in hexane, 200 µg/cartridge), or with 2-heptanone (1:1,000 in paraffin oil, > 98% purity, Carlo-Erba) as positive control. Analysis of EAGs was carried out under pClamp 10 (Molecular Devices). Repolarization rates were compared between the different strains. Repolarization rate was defined as: ((maximum amplitude of depolarization - amplitude of depolarization at the end of stimulation) ÷ maximum amplitude of depolarization) × 100.

## Analyses of T1 sensilla responses by single-sensillum recordings

Single-sensillum recordings were performed as described previously [50] in order to follow the response of T1 sensilla more precisely. In brief, a fly was restrained, a reference electrode was placed in the eye, and the recording tungsten electrode was brought in contact

with the base of a sensillum. Signal was amplified (×1,000, Syntech UN 06, Hilversum, The Netherlands). Actions potentials were analyzed offline with Autospike software (v. 4.0, Syntech). Three doses of cVA were tested, 0.5 µg, 5 µg and 50 µg, and two stimulus durations, 0.5 s and 3 s. Responses from individual ORNs were calculated as the increase in spike frequency relative to the prestimulus frequency. Average firing activities during the stimulus duration, as well as during the 9 s period following the end of stimulus were calculated.

## Courtship assays

Males were isolated after emergence and raised in individual tubes to avoid social interactions. All experiments were done under dim red lights at 25°C (50% to 60% relative humidity) and with immobilized target so as to enhance the behavioral effects of pheromone cues [33]. For male-male assays, a single male (5 to 7 days old) was placed in a test chamber (3 cm diameter, 0.5 cm height) for 10 minutes before introducing a decapitated CS 'target' male. Courtship behavior was observed over 10 minutes and a courtship index (CI) was calculated. CI is the fraction of time spent in courtship activity in the 10-minute observation period. For each tested male, the latency before the first wing vibration and the number of copulation attempts were noted, as an indicator of courtship vigor. Heterosexual courtship was first measured in the same conditions as described for male-male courtship assay, except that a decapitated CS 'target' virgin female was introduced in the test chamber. Synthetic cVA was then applied on the dorsal abdomen of decapitated virgin females before to offer them to mutant or control males. Mature male flies contain approximately 1 µg of cVA in their ejaculatory bulb [51] and 400 ng on their cuticle [52]. The quantity of cVA transferred to the female during copulation was estimated to 200 ng [24] but 70% of the pheromone is lost 6 h after insemination [17]. The amount of cVA on females 24 h after mating was indeed only 10 ng [32]. Three doses of cVA diluted in acetone (> 98% purity, Sigma-Aldrich) were applied on females, 400 ng (twice male equivalent), 150 ng (approximately two-thirds male equivalent) and 50 ng (one-quarter male equivalent).

## Dispersion assay

Experiments were performed as described previously [16]. Briefly, 15 males were raised together after emergence for 5 to 7 days. Six males were simultaneously introduced in a plastic tube (2.5 cm diameter, 10 cm height) coated with Fluon (Sigma-Aldrich) and a small cup containing food was placed in the center, together with a small piece of filter paper containing either 500 µg of synthetic cVA or acetone (solvent). Flies were
videotaped for 30 minutes and the total number of flies on the food cup was counted every 30 s.

## Control of olfactory behavior and of locomotor activity

The response of male flies to airborne chemicals was evaluated using the principle of the olfactory trap assay [34], which allows to test the ability of flies to detect and migrate toward a source of olfactory attractant. All tests were performed under dim red lights as for courtship assays. Ten flies of the same genotype (CS or Est-6° males) were placed in a 50 ml Greiner tube along with a trap constructed from a microfuge tube and two micropipette tips. Traps contained either a fresh yeast paste included in 10% agar as attractant or only agar. The number of trapped flies was counted after 22 h.

To verify that *Est-6°* males were not impaired in their mobility, their locomotor activity was determined as previously described [53]. In brief, a single male was placed in the test chamber containing a filter paper with a bisecting line. The number of times the male crossed the line in a 3-minute observation period was counted.

## Statistical analysis

Analyses were performed with Statistica 7 (StatSoft Inc., Tulsa, OK, USA). The Student's t test or Mann-Whitney U test were used for pairwise comparisons and analysis of variance (ANOVA) followed by a Fisher post-hoc test was performed for comparisons among multiple groups. *P* < 0.05 was accepted as statistically significant.

## Additional material

**Additional file 1: Figure S1.** Dose-response curve for CS male antennae to cis-vaccenyl acetate (cVA), plotted as mean ± SEM; N ≥ 7 for each data point.

**Additional file 2: Figure S2.** Control tests for behavioral analysis. (A) Olfactory trap assay using fresh yeast paste as attractant. (B) Locomotor activity. Mean ± SEM; N = 10 and 40, respectively. Student's t test, \*P < 0.05.

## Acknowledgements

We warmly thank C Coppin for providing the *Est-6* rescue flies, C Wicker-Thomas for the CS strain and BestGene for the transgenic flies. We thank Professor R Vogt for helpful discussions, and M Solvar and Coline Sentenac for technical help. This work was supported by Université Pierre et Marie Curie (UPMC), INRA, CSIC, SLU and a fellowship from the Research Ministry to ND.

## Author details

## Authors' contributions

TC and AF carried out electrophysiological, behavioural and molecular analyses, and participated in interpretation of the results and preparation of the manuscript. GR participated in molecular analyses. PL and TD participated in electrophysiological analyses, interpretation of the results and preparation of the manuscript. MMC conceived of the study, participated in interpretation of the results and wrote the paper.
All authors read and approved the final manuscript.

## Competing interests

The authors declare that they have no competing interests.

Received: 27 February 2012 Accepted: 21 June 2012

Published: 21 June 2012

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doi:10.1186/1741-7007-10-56

Cite this article as: Chertemps et al.: A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in *Drosophila*. *BMC Biology* 2012 10:56.

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