**3. Results**/**Discussion**

We investigated in vivo the involvement of ODEs in the discrimination of sex pheromones in *D. melanogaster*. In this species, female and male pheromones diverge and induce reciprocal e ffects: they tend to stimulate or to inhibit male courtship behavior, respectively [61]. We discovered that a transposable P-element (P-UGT36E1) inserted into the UGT36E1 gene a ffected male discrimination of sex pheromones (Figure 1A). To determine the ability of single tester males to discriminate sex partners, we measured their courtship intensity (or courtship index = CI) toward both female and male target flies simultaneously presented [55]. This paradigm allowed us to measure their CI towards a female target fly (CIf) and towards a male (CIm). The CIf/CIm comparison allowed us to determine the ability of tester males to discriminate the two sex targets, for each genotype and experimental condition. We compared male discrimination ability under red light (Figure 1B; filled bars), in which visual stimuli

provided by both target flies are ineffective and under white light (empty bars) allowing tester males to discriminate sex target based on their different morphology. Under red light, P-UGT36E1 homozygous mutant males showed similar CIm and CIf. In particular, mutant males showed a decreased CIf compared to wild-type tester males (*p* = 0.0045), while their CIm was not affected (*p* = ns). The loss of sex discrimination was likely due to a chemosensory defect given that mutant males tested under white light showed a strong preference to the female target, similarly to wild-type males (Figure 1B). This indicates that the mutation did not affect its overall sexual activity; moreover, the male ability to use visual cues to discriminate sexual partners is functional. Therefore, these results sugges<sup>t</sup> that the P-UGT36E1 mutation affects the ability of male flies to discriminate sex pheromones.

**Figure 1.** Effects of the P-element insertion in UGT36E1. (**A**) Schematic organization of the 37B1 chromosomal region. Arrowhead indicates the position of the P-element inserted in the 5'UTR region of UGT36E1. (**B**) The P-element inserted in UGT36E1 (P-UGT36E1) affects sex pheromone discrimination in male flies. Tests were carried out either under red light (filled bars) or white light (empty bars). Each mirrored bar represents the mean (± s.e.m.) courtship index towards female (CIf) and male (CIm). Individual 4-day-old tester males directed towards female (right) and male (left) headless targets, simultaneously presented during a 5 min observation period. Tester males were homozygous (P-UGT36E1) for the P-element mutation, or wild-type (Dijon strain); target flies always belonged to

this wild-type strain. Significant di fferences for male ability to discriminate between the two sexes are shown next to each mirrored bar as \*\*\*: *p* < 0.001; \*\*: *p* < 0.01; \*: *p* < 0.05 (Student's *t*-test). Courtship data towards each sex were tested using ANOVA and LSD Fisher tests (letters within bars indicates significant di fferences towards each sex). For each test, the number (n) was *n* > 40 (under red light) and *n* > 30 (under white light). ( **C**) The P-element insertion a ffects RNA expression levels of UGT36E1. The expression levels of UGT36E1, UGT36D1 and CG17597 were analyzed by real-time PCR. The significance of differences in the ratio of transcript levels was based on a comparison between wild-type and P-UGT36E1 homozygous mutant flies (in log2 scale control = 0). Data represent the mean (± s.e.m.) of the expression ratio (mutant: wild-type) carried out with three independent extractions.

Given that the P-element inserted upstream of the UGT36E1 gene is also in the vicinity of two other genes (UGT36D1, CG17597; Figure 1A), we measured mRNA expression of the three genes in P-UGT36E1 mutant males. Quantitative RT-PCR (q-PCR) revealed that only UGT36E1 significantly changed its mRNA expression (>two-fold increase in P-UGT36E1 flies as compared to controls; Figure 1C). The absence of any significant variation in expression between the head, thorax, abdomen, and appendages of either wild-type flies or mutant flies suggests that this UGT has not a tissue-specific expression (Figure S1). Moreover, this ubiquitous mutation-induced increase of UGT expression in P-UGT36E1 flies had no general behavioral e ffect given that both locomotor activity and global courtship index (CIf + CIm) were similar to those of wild-type flies under white light (Figure S2; Figure 1B).

To expand our investigation on the behavioral e ffect of UGT36E1, we used RNAi targeted against UGT36E1 (UAS-dsUGT36E1; hereafter dsUGT36E1) to knock down UGT36E1 RNA expression. We targeted dsUGT36E1 distinct subsets of tissues using several GAL4 drivers. We first used the *neuralized*-GAL4 (*neur-*GAL4) driver to target adult chemosensory organs involved in the perception and processing of pheromones (antenna, proboscis, wing margin and tarsa; Figure 2A–C). We found that *neur*-Gal4 is expressed in antennal neuronal cells (Figure 2D,E). Under red light, experimental males ("*neur-*GAL4/+, dsUGT36E1/+") showed increased sex discrimination compared to both control parental transgenic genotypes (*neur-*GAL4/+ and dsUGT36E1/+; Figure 2F). This e ffect was mostly due to the lower CIm shown by knockdown flies (Cim = 6.5 vs. 16.5 for dsUGT36E1/+ control males; *p* = 0.022). However, CIf were similar in mutant and control tester males (CIf = 28 and 32, respectively). Moreover, *neur-*GAL4/+, dsUGT36E1/+ males showed a 6-fold decrease for mRNA expression level in sensory appendages and heads compared to dsUGT36E1/+ controls (*p* = 0.031; Figure 2G). Di fferently, no expression di fference was detected between the abdomen and thorax of the two male genotypes.

**)**

 **\***

**Figure 2.** Effect of dsUGT36E1 in the peripheral chemosensory system. A strong expression of *neuralized*-GAL4 (*neur-*GAL4) was detected in (**A**) the antennae and the proboscis, (**B**) wing margins, (**C**) legs of adult flies. (**D**) Antenna stained for Elav protein (red) and anti-GFP (*neur*-GAL4). (**E**) Magnified view of antennal neurons expressing Elav and *neur*-GAL4. (**F**) Male ability to discriminate sex partners in "*neur-*GAL4/+, dsUGT36E1/+" testers and in both transgenic controls (*neur-*GAL4/+ and dsUGT36E1/+) under red light (filled bars) and white light (empty bars). For each test, *n* > 35. (**G**) Real-time PCR analysis showing UGT36E1 mRNA level in different tissues of "*neur-*GAL4/+, dsUGT36E1/+" flies. The significant difference in transcript level ratio is based on a comparison between control (dsUGT36E1/+) and "*neur-*GAL4/+, dsUGT36E1/+" genotypes (pair-wise fixed reallocation randomization test). For statistics and conditions, see Figure 1. \*\*\* *p* < 0.001; \*\* *p* < 0.01; \* *p* < 0.05.

Next, we targeted subsets of peripheral chemosensory neurons potentially involved in pheromonal perception. Given that "*neur-*GAL4/+, dsUGT36E1/+" males showed reduced CIm, we targeted the dsUGT36E1 transgene in Gr66a gustatory sensory neurons which are involved in the detection of a male aversive pheromone [62,63]. The CIf/CIm performance of transgenic males was not different compared to controls (Figure S3), indicating that UGT36E1 expression in Gr66a-expressing neurons is not required for sex discrimination. Differently, when dsUGT36E1 was targeted in the majority of peripheral olfactory sensory neurons (OSNs) using the *Orco*-GAL4 driver, manipulated males ("*Orco*-GAL4/+, dsUGT36E1/+") showed a higher discrimination ability as compared to controls (Figure 3A). This effect was due both to (i) the increased CIf of manipulated males compared to *Orco*-Gal4/+ control (*p* = 0.0003) and to (ii) their decreased CIm compared to dsUGT36E1/+ males (*p* = 0.0034). Note that *Orco*-Gal4/+ control transgenic males, which showed a wild-type-like discrimination, had a significantly

decreased sexual activity to target females and/or to target males compared to several other control transgenic males (Figure S4). While the potential alteration induced by GAL4 in some chemosensory tissues has already been reported [62], this finding supports the idea that male sexual activity and sex discrimination can be affected separately. The role of UGT36E1 in sex pheromones detection provides also the first functional evidence of the involvement of an ODE in chemosensory neurons. We cannot exclude the possibility that non-neuronal accessory cells which also express ODEs [64,65] can additionally modulate the male sex pheromone(s) perception. Moreover, our data only provides an indirect evidence of the UGT36E1 expression in head olfactory appendages since we obtained no signal using an antibody specifically designed against this protein.

**Figure 3.** Expression and effect of dsUGT36E1 targeted in various sensory neurons subsets. (**A**) Targeting the dsUGT36E1 transgene in most olfactory sensory neurons with the *Orco*-GAL4 transgene ("*Orco*-GAL4/+, dsUGT36E1/+") improved mate choice performance compared to both transgenic controls. A similar targeting of this transgene in the P-UGT36E1 mutant background (in "*Orco*-GAL4/+, dsUGT36E1/+", but not "P- UGT36E1/+, dsUGT36E1/+") rescued male performance. For each behavioral test, *n* > 35. All courtship tests were carried out under red light. (**B**) Quantitative analysis of UGT36E1 expression in the sensory appendages of "P-UGT36E1/+, *Orco*-GAL4/+, dsUGT36E1/+" flies compared to "*Orco*-GAL4/+, dsUGT36E1/+" and "P-UGT36E1/+, dsUGT36E1/+" control flies. The wild-type strain Dijon was used as a reference. For statistics and conditions, see Figure 1. \*\*\* *p* < 0.001; \*\* *p* < 0.01; \* *p* < 0.05.

Given the reciprocal e ffects induced by the P-UGT36E1 mutation and by the dsUGT36E1 RNAi transgene, both on UGT36E1 mRNA level and sexual discrimination, we combined the two genetic tools in the same fly. Strikingly, "P-UGT36E1/+, *Orco*-GAL4/+, dsUGT36E1/+" males showed a wild- type-like discrimination ability, whereas control "P-UGT36E1/+, dsUGT36E1/+" males showed no such preference (Figure 3A). This indicates that the dsUGT36E1 RNAi (increasing sex discrimination) compensated for the behavioral defect caused by the P-UGT36E1 mutation (decreasing sex discrimination). Moreover, UGT36E1 mRNA levels, measured in the fly appendages, did not di ffer between "P-UGT36E1/+, *Orco*-GAL4/+, dsUGT36E1/+" males, on one hand and wild-type and transgenic control flies on the other (Figure 3B). These experiments strongly sugges<sup>t</sup> that the gene mutation and the RNAi transgene have additive e ffects both at the molecular and behavioral levels.

If we assume that both mRNA and UGT enzyme levels are correlated, our data sugges<sup>t</sup> that the ability of male flies to discriminate sex pheromones depends on the UGT expression level in antennal OSNs. Compared to wild-type males, a reduction of UGT expression level in OSNs tends to increase male ability to discriminate sex pheromones, while a higher level induces the opposite effect. This suggests that the expression level of the UGT gene product in wild-type flies is somewhat intermediate between the levels in the P-UGT36E1 mutant and in "*Orco*-GAL4/+, dsUGT36E1/+" males. Such intermediate level may reflect a trade-o ff between a relatively high non-specific expression for optimal detoxification and/or signal termination and a relatively low expression allowing wild-type male to acutely detect and discriminate pheromonal stimuli.

To further investigate the function of UGT36E1 in the peripheral olfaction of sex pheromones, we recorded the global electrophysiological responses of individual male fly antennae (electroantennogram = EAG) either stimulated by male or female volatile pheromonal compounds. We used EAG instead of single sensilla recordings (SSRs) given that we had no idea of the sensillum or sensilla which could respond to the pheromonal mixture. Responses to sex-specific stimuli were normalized with 2-H, a general odorant eliciting robust antennal responses. The responses to these three olfactory stimuli were compared between wild-type, P-UGT36E1 mutant and "*Orco*-GAL4/+, dsUGT36E1/+" males (and in their transgenic parental controls). Wild-type male antenna showed slightly larger relative responses to male than to female volatile compounds (*p* = 0.014; Figure 4A,B). In P-UGT36E1 mutant males, the di fference of relative responses to male and female volatile compounds strongly increased (*p* = 0.0006). This increased di fference was due to a significantly increased response to male volatile compounds in the mutant compared to wild-type males (*n* = 11–15; *p* = 0.02). However, both mutant and wild-type males showed similar relative responses to female volatile compounds. On the other hand, "*Orco*-GAL4/+, dsUGT36E1/+" experimental males showed similar relative responses to female and male volatile compounds whereas parental transgenic controls (dsUGT36E1/+ and *Orco*-GAL4/+) showed a wild-type-like pattern (e.g., a slightly larger relative response to male than to female volatile compounds; Figure 4C,D). These results indicate that the P-UGT36E1 mutation enhanced the relative amplitude of the electrophysiological response to male volatile compounds whereas the RNAi directed against UGT36E1 in OSNs reduced this response. Therefore, if we cannot formally rule out the possibility that the UGT also a ffected the EAG response to 2-H, our data clearly show that the manipulation of the UGT gene a ffected the EAG response to male pheromone(s) relative to female pheromone(s).

**Figure 4.** Electrophysiological recording of male olfactory response to different stimuli. (**A**) The graphs represent averaged electroantennogram (EAG) responses of wild-type and P-UGT36E1 mutant male flies to three stimuli: living females, living males and 2-Heptanone (2-H). The thick bars indicate the stimulus duration. (**B**) Bars represent the EAG responses to males and females normalized with the respective responses to 2-H. Statistical differences were noted (i) for the significance of the difference to both sex stimuli (above each pair of bars) and (ii) for responses to male stimuli (letters inside lightly filled bars), between genotypes. (**C**) Averaged EAG responses for "*Orco*-GAL4/+, dsUGT36E1/+", "dsUGT36E1/+" and "*Orco*-GAL4/+" males presented with the same three stimuli. (**D**) Normalized EAG responses to males and females. "*Orco*-GAL4/+, dsUGT36E1/+" males showed no difference in their responses to either sex. For each test, *n* > 11. For statistics and conditions, see Figure 1. \*\*\* *p* < 0.001; \*\* *p* < 0.01; \* *p* < 0.05.

Involvement of ODEs in pheromonal signal modulation was previously hypothesized [20,35,36, 66–68]. Here we propose that the abundance of the UGT36E1 enzyme in *Drosophila* OSNs can affect the clearance of pheromones in the perireceptor space. Based on this hypothesis, the over-expression of the gene (in P-UGT36E1 mutants) would increase the clearance of the stimulus, promoting faster successive stimulations, and thus enhance the effect of the male inhibitory pheromone (as measured by the EAG), this leading to reduced sex discrimination. Reciprocally, the decreased UGT expression in RNAi targeted males could affect the pheromonal clearance, resulting in an overstimulation which negatively impacts the signal level. This would reduce the aversive effect induced by the male pheromone(s) and increase male ability to discriminate sex pheromones. Based on these observations, we hypothesize that decreased pheromonal clearance in the perireceptor space promoted the saturation of dedicated receptors and reduced both OSN sensitivity and relative EAG amplitude. In support of our interpretation, two other studies based on the detection of different chemical or use of different genetic tools, reported that the alteration of activity in phase I enzymes (CYP or CES) also induced a prolonged olfactory neuronal response leading to an altered perception of the pheromones highlighting their role in signal termination [7,36].

How can we explain the apparent conundrum between the increased EAG response to male pheromone and the decreased discrimination shown by mutant males (and the reciprocal effects in RNAi targeted males)? In our behavioral assay, wild-type male OSNs were simultaneously stimulated by a mixture of inhibitory and attractive olfactory pheromones emitted by male and female flies, respectively. We hypothesize that the increased nervous antennal response to male inhibitory pheromone(s) in the perireceptor space may disturb the response to female pheromone(s), and this unbalanced effect would affect their integrated comparison in brain structures which are normally involved in the sex pheromone discrimination [69,70]. In any case, our data reveal that this mechanism likely depends on the level of the UGT gene product: males combining both the mutation and the RNAi targeted in neural tissues showed a wild-type-like behavioral discrimination. This is reminiscent of a recent transcriptomic study performed in the *Bombyx mori* silkworm antenna which revealed that the olfactory impairment observed in the domestic strain is correlated with a decreased expression of ODEs (including some UGTs) as compared to the wild *B. mori* strain [15].

In summary, our data reveal that the reciprocal variation of a UGT expression can change male sex discrimination in opposite directions. This effect is likely based on the ability of manipulated flies to discriminate between volatile sex pheromones. Given the high diversity, the ubiquitous distribution, the regulation and the varied properties of ODEs in animals, our findings provide a significant step to unravel the complexity of mechanisms underlying olfactory sensitivity at the peripheral nervous system.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/3/237/s1, Figure S1: Expression ratio of UGT36E1 in appendages, head and thorax and abdomen, Figure S2: Locomotor activity tests, Figure S3: Effect of dsUGT36E1 targeted in Gr66a gustatory neurons, Figure S4: Male courtship in control strains.

**Author Contributions:** Conceptualization, Y.A., J.-F.F. and J.-M.H.; methodology, P.L., I.C., P.F., F.N., S.F. and A.L.; validation, J.-F.F. and J.-M.H.; formal analysis, S.F., A.L. and P.L.; investigation, S.F., A.L., P.L., P.F. and I.C.; writing—original draft preparation, S.F., A.L., Y.A., L.B., F.N., J.-M.H. and J.-F.F.; writing—review and editing, S.F., A.L., Y.A., L.B., F.N., J.-M.H. and J.-F.F.; supervision, J.-F.F. and J.-M.H.; project administration, J.-M.H.; funding acquisition, Y.A., L.B., J.-F.F. and J.-M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Agence National de la Recherche (ANR-08-BLAN-0203) by the CNRS, INRA, the Burgundy Regional Council and the French Ministry of Higher Education, Research and Innovation.

**Acknowledgments:** We thank Matthew Cobb for comments on a previous version of the manuscript, Catherine Méart and Adrien François for their invaluable technical assistance and DIMACell plateform for microscopy studies (Université de Bourgogne Franche-Comté).

**Conflicts of Interest:** The authors declare no conflict of interest.
