**1. Introduction**

The initiation of olfaction is mediated by olfactory receptors which interact with a variety of odorant molecules allowing their fine detection and discrimination. Receptor activation triggers the olfactory signal transmitted as a depolarization train of spikes to the central nervous system, this resulting in its integrated perception and an adapted behavioral response. The detection of odorants is modulated by perireceptor events through which a complex series of biochemical processes are carried out to influence the entry, exit and/or residence time of odorant molecules in the receptor environment [1,2]. Perireceptor mechanisms play a significant role in the modulation of the stimulus

availability for receptors, therefore impacting its reception and subsequent perception. They include, first, the binding and transport of hydrophobic odorant molecules by odorant-binding proteins (OBPs) [3–5] and then their inactivation and elimination by detoxification enzymes called odorantmetabolizing enzymes in vertebrate (OMEs) [6] or odorant-degrading enzymes (ODEs) in insect [2,7]. Indeed, some metabolizing enzymes can be expressed, either specifically, and/or at a high concentration, in the olfactory tissues or organs [6,8–11]. These enzymes are primarily involved in detoxification processes by catalyzing the biotransformation of hydrophobic xenobiotic molecules through two phases often but not necessarily successive. During phase I, functionalization enzymes introduce, or unmask, functional groups into xenobiotics through oxidation, reduction or hydrolysis reaction (e.g., cytochrome P450, CYP; aldehyde dehydrogenase; carboxylesterase, CES). During phase II, functionalized metabolites can be conjugated to hydrophilic products by transferases enzymes such as UDP-glycosyltransferases (UGT) or glutathione transferases (GST). Thus, the resulting inactive hydrophilic metabolites can be easily eliminated. These enzymes are organized in networks allowing to metabolize a broad range of substrates.

In insects, the characterization of metabolizing enzymes received an increasing interest with regard to their role in insecticide resistance, adaptation to host plant volatile and their function; as ODEs, in the termination of the olfactory signal to maintain a relatively high olfactory sensitivity toward new stimuli. In particular, recent studies investigating the antennal transcriptome in di fferent species identified varied ODEs including CYP, CES, GST and UGT [11–24]. These reports have completed and confirmed the case-by-case identification of previously characterized ODEs [25–31]. Altogether, a high number of diverse antennal ODEs have been identified including among others, 30 CES and 84 CYP in *Spodoptera littoralis* [14,26,27,32,33], and 31 GST and 57 CYP in *Drosophila melanogaster* antennae [11].

However, data about ODE function in chemosensory process are still limited, likely because of (i) the current focus on odorant/receptor interaction and (ii) the complexity due to the high diversity of enzymes and odorant substrates in di fferent species and strains. Phase I ODE's function was initially investigated with regard to the perception of pheromones [2,34,35] because of their critical role and their ability to trigger specific and measurable sexual behavior [7,36]. Although phase II enzymes are expected to play a similar role as phase I enzymes in the metabolism of plant volatiles, pheromones or diverse odorants [9,16,20,22], no olfactory function was revealed so far in insects. Among phase II ODEs, the study of UGTs received an increasing interest during the last decade, especially with regard to insecticide resistance and plant defense mechanisms [37–44]. This major class of enzymes in the animal kingdom [45,46] catalyzes in insects the conjugation of a glycosyl group brought by a UDP-glycoside to hydrophobic substrates [47,48]. The fact that UGT expression is enriched in antennae supports its potential role in olfaction [11,47,49–51], in relation with its high number of isoforms: 20 in *Holotrichia parallela Motschulsk*y and 11 in *S. littoralis* and 19 in *D. melanogaster* [11].

In the present study, we combined molecular, genetic, behavioral and electrophysiological approaches to investigate the influence of a phase II ODE, a UGT (UGT36E1), on the ability of *D. melanogaster* males to discriminate sex pheromones. UGT36E1 (CG17322) is one of the most expressed UGT in *D. melanogaster* [47]. We used both mutational and interferential RNA approaches to target UGT36E1 expression in the fly olfactory tissues. We found that both the decreased and increased UGT expression in the peripheral olfactory system a ffected sex pheromone discrimination in a reciprocal manner.

#### **2. Materials and Methods**

#### *2.1. Stocks and Flies*

All *D. melanogaster* strains were raised on yeast/cornmeal/agar medium and kept at 24 ± 0.5 ◦C with 65 ± 5% humidity on a 12L:12D cycle. The wild-type Dijon2000 strain (Di2), used as a control, has been maintained in our lab for more than a decade. The P-UGT36E1 mutant strain (y1 w67c23); P{SUPor-P}CG17322KG04070 (#13518) and the transgenic strains *neur*-GAL4 (#6393); *Orco*-GAL4

(#23292); UAS-CD8::GFP (#5130) were obtained from the Bloomington Drosophila Stock Center (Indiana University). The *Gr66a*-GAL4 strain was kindly provided by Dr. Hubert Amrein. RNAi transgenics against UGT36E1 were purchased from the VDRC [52]. To isogenize its genetic background, the P-UGT36E1 mutation was outcrossed to the Di2 wild-type genetic background of the w<sup>1118</sup> strain by five successive backcrosses [53]. Crosses were performed using standard techniques and genetic methods [54].
