The Human Odorant Receptor OR10A6 Is Tuned to the Pheromone of the Commensal Fruit Fly Drosophila Melanogaster

bioRxiv preprint doi: https://doi.org/10.1101/2020.12.07.414714; this version posted December 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 The human odorant receptor OR10A6 is 2 tuned to the pheromone of the commensal 3 fruit fly Drosophila melanogaster

4 Tim Frey* 1, Charles A. Kwadha* 2, Erika A. Wallin 3, Elsa Holgersson 4, Erik 5 Hedenström 3, Björn Bohman 2, Marie Bengtsson 2, Paul G. Becher 2, Dietmar 6 Krautwurst 1, Peter Witzgall 2

7 1 Leibniz-Institut für Lebensmittel-Systembiologie an der Technischen Universität 8 München, Lise-Meitner Strasse 34, D-85354 Freising, Germany

9 2 Department of Plant Protection Biology, Swedish University of Agricultural Sciences, 10 Box 102, 23053 Alnarp, Sweden

11 3 Department of Chemical Engineering, Mid Sweden University, Holmgatan 10, 85170 12 Sundsvall, Sweden

13 4 Systembolaget AB, 103 84 Stockholm, Sweden

14 Tim Frey - [email protected] 15 Charles Kwadha - [email protected] 16 Erika A. Wallin - [email protected] 17 Elsa.Holgersson - [email protected] 18 Erik Hedenström - [email protected] 19 Björn Bohman - [email protected] 20 Marie Bengtsson - [email protected] 21 Paul G. Becher - [email protected] 22 Dietmar Krautwurst - [email protected]

23 Correspondence: Peter Witzgall - [email protected]

24 Abstract

25 Background: All living things speak chemical. The challenge is to discover the 26 vocabulary, the volatile odorant chemicals that enable communication across phylogenies 27 and to translate them to physiological, behavioural and ecological function. Olfactory 28 receptors (ORs) interface animals with airborne odorants. Expression of single ORs in 29 human embryonic kidney cells (HEK-293) makes it possible to interrogate ORs with 30 synthetic chemicals and to identify cognate ligands that convey olfactory information. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.07.414714; this version posted December 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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31 Results: The cosmopolitan strain of the vinegar fly Drosophila melanogaster has 32 accompanied the human expansion out of Africa, more than ten thousand years ago. 33 These flies are strictly anthropophilic and depend on human resources and housing for 34 survival, particularly in colder climate zones. Curiously, humans sense the scent of a 35 single fly, and more precisely the female pheromone (Z)-4 undecenal (Z4-11Al), at 10 36 ng/mL (0.06 µmol/L). A screening of all functional human ORs in a HEK-293 assay 37 provides an explanation for this astounding sensitivity, as it shows that OR10A6, one of 38 the most highly expressed human ORs, is specifically tuned to Z4-11Al. Chemical analysis 39 of fly effluvia confirms that cosmopolitan D. melanogaster females release Z4-11Al, while 40 females of an African fly strain from Zimbabwe release a 1:3-blend of Z4-11Al and (Z)-4 41 nonenal (Z4-9Al). Interestingly, a blend of Z4-9Al and Z4-11Al produces a different 42 aroma than the the single compounds, which is why we readily differentiate cosmopolitan 43 and Zimbabwe flies by nose.

44 Conclusion: That we sensitively and specifically perceive the fly pheromone Z4-11Al 45 suggests that it is a component of human odour scenes. This may have afforded a 46 sensory drive during adaptation of commensal flies to human habitats and selected for a 47 role of Z4-11Al in fly aggregation and premating communication. Screening ORs for key 48 ligands leads to the discovery of messenger chemicals that enable chemical 49 communication among and betwen vertebrate and invertebrate animals.

50 Keywords

51 pheromone, semiochemical, food odour, aroma, odorant receptor, olfaction, Drosophila

52 Background

53 Volatiles from animals, microorganisms and plants are yielded by basic metabolic 54 pathways, they inform about resources and habitats, and reveal identities and social 55 context. Communication via volatile chemicals is reliable and inclusive, for all living 56 beings across the kingdoms.

57 Volatiles are aired tweets for those equipped with receptors and sensory circuits to 58 capture and interpret them. Animals possess olfactory receptors (ORs) for peripheral 59 detection of volatiles, and for filtering out scents that make ecological and behavioural 60 sense, from a noisy chemical airspace. The importance of ORs in interfacing animals with 61 the chemical environment is reflected by the strong selection pressure on ORs to adapt to 62 ecosystem and habitat cues and to social and sexual signals (Hayden et al. 2010, 63 Fleischer et al. 2018, Robertson 2019, Saraiva et al. 2019). Olfaction has developed 64 independently in invertebrates and vertebrates, but the overarching organisation of the

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65 olfactory system, building on rapidly evolving ORs, expressed in peripheral olfactory 66 sensory neurons, feeding into a hierachy of central olfactory circuits, is rather similar in 67 all animals (Su et al. 2009, Bear et al. 2016)

68 A principal, current objective and fascinating challenge in vertebrate and invertebrate 69 olfaction research is to explore the receptive range of ORs and to set landmarks in 70 chemical space, for a comprehension of olfactory codes and the functional analysis of 71 olfactory systems. Expression of ORs in heterologous cell systems, for example in human 72 embryonic kidney (HEK-293) cells, enables experimental access and makes it possible to 73 interrogate individual ORs and to identify their cognate ligands (Krautwurst et al. 1998, 74 Corcoran et al. 2014). ORs are seven-transmembrane domain G-protein coupled 75 receptors (GPCRs) and a first challenge is to achieve fully functional membrane 76 expression (Noe et al. 2017b). The ensuing step is to compose comprehensive and 77 manageable, yet representative panels of biologically relevant compounds for 78 investigating their receptive range. One strategy is to use key food volatiles, identified 79 from the odorant space of food and beverages (Krautwurst & Kotthoff 2013, Dunkel et al. 80 2014). Odorant panels will, however, always remain notoriously incomplete - in 81 comparison with an overwhelmingly diverse odorscape, containing countless chemicals. 82 In vitro OR screenings do afford active ligands, but most human ORs remain in the 83 orphan state (de March et al. 2015, Block 2018).

84 Chemicals we perceive at very small amounts, and which are not strictly associated with 85 food, are particularly inspiring targets for OR screenings. One such candidate compound 86 is (Z)-4-undecenal (Z4-11Al), the volatile female pheromone of the commensal fruit fly 87 Drosophila melanogaster. In Drosophila, two isoforms of DmelOR69a, with a dual 88 specificity for food odorants and pheromone, are co-expressed in the same OSNs. 89 Intriguingly, we ourselves readily perceive Z4-11Al, which is released at subnanogram 90 amounts per hour, and we reliably distinguish between the sexes, since only female flies 91 produce this scent (Lebreton et al. 2017, Becher et al. 2018).

92 Cosmopolitan D. melanogaster flies are strictly anthropophilic, since they accompanied 93 the human expansion from out of Africa more than 10.000 ya, and they are partially 94 reproductively isolated from African flies, such as the Zimbabwe strain (Lachaise & Silvain 95 2004). The cosmopolitan fly pheromone Z4-11Al is an oxidation product of the 96 aphrodisiac cuticular hydrocarbon (Z,Z)-7,11-heptacosadiene (7,11-HD) (Billeter et al. 97 2009, Lebreton et al. 2017). Since females from Zimbabwe produce more (Z,Z)-5,9- 98 heptacosadiene (5,9-HD) than 7,11-HD (Dallerac et al. 2000, Grillet et al. 2012), it 99 follows that these flies would release another aldehyde. Posing that our perception of Z4- 100 11Al is not only sensitive but also specific, we asked whether we are able to olfactorily 101 discriminate between females of the cosmopolitan and Zimbabwe strains of D. 102 melanogaster. A sensory panel corroborated this idea by comparing synthetic compounds 103 and fly odours.

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104 Naturally, this begs the question - how do humans smell the scent of the fly? A range of 105 human ORs is tuned to straight-chain aldehydes, which are commonly found in fruit and 106 vegetable aroma (Schmiedeberg et al. 2007, Saraiva et al. 2009, Nara et al. 2011, Li et 107 al. 2014, de March et al. 2015, Block et al. 2018) and perception of Z4-11Al might be 108 encoded by one or even several of these aldehyde-responsive ORs. We hence submitted 109 Z4-11Al to an in vitro screening of all functional human ORs, using heterologous 110 expression in human embryonic kidney cells (HEK-293) and a luminescence-based assay 111 (Noe et al. 2017a,b). This screening renders OR10A6 as the sole responsive OR for Z4- 112 11Al. A subsequent olfactory panel test confirmed the results of an in vitro dose-response 113 test of several synthetic aldehyde analogues, showing that we discriminate between 114 structurally related aldehydes and that our olfactory perception of Z4-11Al is remarkably 115 sensitive and specific.

116 The scent of the fly illustrates how chemical ecology research inspires the discovery of OR 117 ligands and provides another account for chemical communication across phylogenies.

118 Materials and methods

119 Chemicals

120 Isomeric purity of (Z)-4-undecenal (Z4-11Al) was 98.6%, according to gas 121 chromatography coupled to mass spectrometry (6890 GC and 5975 MS, Agilent 122 Technologies, Santa Clara, CA, USA). Isomeric purity of (Z)-4-nonenal (Z4-9Al) and (Z)- 123 6-undecenal (Z6-11Al) were 97.4% and 96%, repectively. Chemical purity of these 124 synthetic aldehydes was >99.9%. Ethanol (redistilled; Merck, Darmstadt, Germany) was 125 used as solvent.

126 For the OR screening assays, the following chemicals were used: Dulbecco’s MEM medium 127 (#F0435), FBS superior (#S0615), L-glutamine (#K0282), penicillin (10000 128 U/ml)/streptomycin (10000 µg/ml) (#A2212), trypsin/EDTA solution (#L2143) 129 (Biochrom, Berlin, Germany), CaCl2*2H2O (#22322.295), D-glucose (#101174Y), 130 dimethyl sulfoxide (DMSO) (#83673.230), HEPES (#441476L), potassium chloride 131 (#26764.230), and sodium hydroxide (#28244.295) (VWR Chemicals BDH Prolabo, 132 Leuven, Belgium), sodium chloride (#1064041000, Merck, Darmstadt, Germany), 133 ViaFect™ Transfection Reagent (#E4981, Promega, Walldorf, Germany), D-luciferin 134 (beetle) monosodium salt (#E464X, Promega, Walldorf, Germany), Pluronic® PE 10500 135 (#500053867, BASF, Ludwigshafen, Germany), (R)-(-)-carvone (#W224908, Sigma- 136 Aldrich, Steinheim, Germany).

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137 Insects

138 Cosmopolitan (Dalby) and Zimbabwe (S-29, Bloomington) strains of D. melanogaster 139 were reared on a standard sugar-yeast-cornmeal diet at room temperature (25±2°C) and 140 50±5 rH under a 12:12-h L:D photoperiod. Eclosing flies were collected every 4 h and

141 sexed under CO2, according to the sex comb on the third segment of the male forelegs. 142 Presence of meconium was used as a distinguishing feature for virgin flies. Females were 143 kept separately in 30-mL Plexiglas vials with fresh food.

144 Pheromone collection and chemical analysis

145 Sixty unmated cosmopolitan and Zimbabwe females, respectively (n=9 and n=10, 146 respectively) were transferred to standard glass rearing vial (24.5 x 95 mm, borosilicate 147 glass; Fisher Scientific, Sweden), which had been baked at 350°C overnight. After 24 h, 148 the flies were removed and the vial was rinsed with 200 µl of hexane, containing 100 ng 149 decanal as internal standard, in an ultrasonic water bath for 3 min. The solvent was 150 transferred to 1.5 mL GC-MS vials with insert and condensed to ca. 5 µl in a fume hood.

151 Two µl of the solvent rinses were analyzed by gas chromatography-mass spectrometry 152 (GC-MS) (6890 GC and 5975 MS, Agilent, Santa Clara, CA, USA) on a fused silica

153 capillary column (60 m x 0.25 mm), coated with HP-5MS UI (df =0.25 µm; Agilent). 154 Injections were made in splitless mode (30 s), at 275°C injector temperature. The GC 155 oven was programmed from 50 to 250°C at 8°C/min (2 min and 10 min hold, 156 respectively) and a final temperature of 275°C, the mobile phase was helium (34 cm/s). 157 The MS operated in scanning mode. Aldehydes were identified according to m/z spectra 158 and Kovats retention indices, using custom and NIST libraries, in comparison with 159 synthetic standards.

160 OR expression and screening

161 Cell culture and transient DNA transfection

162 Human embryonic kidney (HEK-293) cells were cultivated in Dulbecco’s MEM medium 163 (DMEM: w 3.7 g/L NaHCO3, w 4.5 g/L D-glucose, w/o L-glutamine, w/o Na-pyruvate) 164 supplemented with 10% fetal bovine serum (FBS superior), 2 mM L-glutamine, 100 165 units/mL penicillin, and 100 µg/mL streptomycin in 10 cm cell culture dishes at 37 °C, 166 5% CO2, and 100% humidity, as test cell systems for the functional expression of 167 recombinant ORs as described previously (Geithe et al. 2015, 2017b; Noe et al. 2017a). 168 One day before transfection, HEK-293 cells were transferred with a density of 12000 cells 169 per well in white 96-well plates (Thermo Scientific™ Nunc™ F96 MicroWell™, white, 170 #136102, Thermo Fisher Scientific, Waltham, USA). The transfection was done by the 171 cationic lipid-transfection method using 100 ng OR plasmid-DNA, 50 ng olfactory G- 172 protein Gαolf (Jones & Reed 1989, Shirokova et al. 2005), 50 ng RTP1S (Saito et al.,

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173 2004), 50 ng Gγ13 (Li et al. 2013), and 50 ng genetically modified cAMP-luciferase 174 pGloSensor™-22F (Binkowski et al. 2009) (Promega, Madison, USA), each with ViaFect™ 175 Transfection Reagent. As negative control, transfection of an empty pFN210A-vector- 176 plasmid (mock) was employed. As positive control OR1A1 was transfected on each plate. 177 Each transfection was done in triplicates on the same 96-well plate. The cells were taken 178 into experiment 42 h post-transfection as reported (Geithe et al. 2015, 2017b; Noe et al. 179 2017a).

180 cAMP luminescence assay

181 Cell culture media of the transfected HEK-293 cells in the 96-well plates was replaced 1 h 182 prior to the luminescence measurement with physiological salt solution containing 140 183 mmol/L NaCl, 10 mmol/L HEPES, 5 mmol/L KCl, 1 mmol/L CaCl2, 10 mmol/L D-glucose 184 and 2% D-luciferin, pH 7.4. After this incubation, basal luminescence signals for each well 185 (three consecutive data points, 60 s intervals) were recorded with the GloMax® Discover 186 Microplate Reader (Promega, Madison, USA) before odorant application. As positive 187 control, 30 µmol/L (R)-(-)-Carvone was applied on the OR1A1 transfected cells. Odorant 188 stock solutions were prepared in DMSO, and diluted 1:1000 into the physiological salt 189 solution containing 0.05% Pluronic® PE 10500, as solvent mediator. Final DMSO 190 concentration on the cells was 0.1%. 4 min after odorants were applied to the cells, three 191 consecutive data points at 60 s intervals were recorded for each well with the GloMax® 192 Discover Microplate Reader.

193 Data analysis of cAMP luminescence measurements

194 The raw luminescence data obtained from the GloMax® Discover Microplate Reader were 195 processed as followed. For each well, the average of the three data points before odorant 196 addition was subtracted from the average of the three data points after stimulation 197 (Δsignal). Then, the corresponding mock of each substance/concentration was subtracted 198 from each Δsignal value. All mock-subtracted Δsignal-value were then normalized to the 199 positive control of each plate (screening experiments), or to the respective maximum

200 signal of each concentration-response relation. EC50 values were obtained by fitting the 201 function f = ((a-d)/(1+(x/EC50)^h)+d) to the data.

202 Sensory evaluation

203 Synthetic compounds were diluted in redistilled ethanol (Sigma-Aldrich). One h prior to 204 testing, 10-µL aliquots were pipetted into 20-mL screw-top glass vials (Genetec) 205 containing 10 mL of redistilled water or 10 mL of white wine (Ruppertsberger Riesling, 206 Systembolaget, 72038-01). For evaluation of fly odour, 5 live D. melanogaster females, 207 of the cosmopolitan (Dalby) and Zimbabwe strain (S-29, Bloomington), respectively, 208 were placed during 3 h in 20-mL vials, they were released ca 30 min before testing, and 209 10 mL of redistilled water or wine was added to the vial.

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210 A first pairwise comparison comprised vials containing either 10 ng synthetic Z4-11Al or 211 fly odour, in 10 mL water or wine. Vials were assigned a random letter. Panelists (n=21) 212 were asked whether or not the odours in the vials bear resemblance to each other. 213 Results were compared using a paired Student's t-test.

214 Subsequent triangle tests included (a) vials exposed to cosmopolitan and Zimbabwe D. 215 melanogaster female flies (n=45 judges), (b) 10 ng Z4-9Al or 10 ng Z4-11Al (n=45), (c) 216 a blend of 10 ng Z4-9Al and 3 ng Z4-11Al (n=41), and (d) and a dose-response test, of 6 217 different amounts of synthetic Z4-9Al, Z4-11Al and Z6-11Al, from 0.01 ng to 10 ng 218 (n=9), in 10 mL water. Of three stimuli in each triangle, two were the same and judges 219 were asked to point out the odd sample. Sample order and lettering of vials were 220 randomized, a Chi2-test was used for statistical comparison.

221 Results

222 Sensory evaluation of fly odour vs Z4-11Al

223 A sensory panel compared synthetic Z4-11Al with the scent of D. melanogaster females, 224 of the cosmopolitan and the Zimbabwe strains, in water and wine, providing a rich 225 odorant background (Figure 1).

226 Vials, where 5 fly females had been kept during 3 h and released 30 min before testing, 227 and vials formulated with 10 ng Z4-11Al, were filled with 10 mL water or wine, 228 respectively. In vials filled with water, a significant number of panelists found the odour 229 of Z4-11Al to resemble cosmopolitan flies (P=0.0002), but not Zimbabwe flies (P=0.002; 230 Figure 1). In wine, 19 out of 21 panelists readily perceived cosmopolitan fly odour and 231 found it to resemble synthetic Z4-11Al (P<0.0001). In contrast, the evaluation of 232 Zimbabwe fly odour vs. Z4-11Al was impaired in wine. The number of panelists who 233 distinguished and did not distinguish Z4-11Al from Zimbabwe flies was not different at 234 P>0.05 (Figure 1).

235 Aldehyde emission by cosmopolitan and African flies

236 Cosmopolitan D. melanogaster females produce the courtship pheromone 7,11-HD, which 237 affords Z4-11Al as oxidation product (Billeter et al. 2009, Lebreton et al. 2017). Females 238 of the Zimbabwe strain produce mainly 5,9-HD instead (Dallerac et al. 2000), and it 239 follows that these flies would therefore also release Z4-9Al (Figure 2A).

240 Headspace analysis of D. melanogaster confirmed that females of the cosmopolitan strain 241 released Z4-11Al, while Z4-9Al remained below detection level in all fly effluvia

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242 collections. Zimbabwe females, on the other hand, produced Z4-9Al, in addition to Z4- 243 11Al, in a 2.6±0.7:1 ratio amount (n=10) (Figure 2B).

244 The human olfactory receptor OR10A6 is tuned to Z4-11Al

245 OR10A6 L287P showed by far the strongest response to Z4-11Al, in a screening of 579 246 human ORs expressed in human embryonic kidney cells (HEK-293) (Figure 3). A dose-

247 response assay confirmed that Z4-11Al was a most active ligand for OR10A6 L287P, 248 compared with the aldehyde analogue Z4-9Al and the positional isomer Z6-11Al (Figure 249 4A).

250 The odorant panel corroborated that we are more sensitive to Z4-11Al than to Z4-9Al or 251 Z6-11Al (Figure 4B). A significant number of panelists sensed Z4-11Al at 10 ng/mL in 252 water (0.06 µmol/L). In comparison, panelists did not perceive Z4-9Al or Z6-11Al, at the 253 amounts tested.

254 A low response threshold to Z4-11Al, in vitro (Figure 4A) and in vivo (Figure 4B) 255 corroborates our remarkable sensitivity to the female pheromone of cosmopolitan D. 256 melanogaster. Panelists who discriminated Z4-11Al from control at the higest dose 257 (Figure 4B) associated its aroma with citrus or grapefruit. In comparison, the aroma of 258 Z4-10Al is reminiscent of tangerine (Douglas et al. 2001).

259 Specific perception of Z4-11Al

260 In a subsequent triangle test, panelists discriminated between cosmopolitan and 261 Zimbabwe female flies (P=0.01; Figure 5A). Discrimination between Z4-9Al and Z4-11Al 262 (P=0.02; Figure 5B), however, cannot account for this observation. Emission of a blend of 263 Z4-11Al and Z4-9Al, by Zimbabwe strain females (Figure 2), together with our far higher 264 sensitivity to Z4-11Al than to Z4-9Al (Figure 4), seemingly contradicts a sensory 265 differentiation between Z4-11Al and Zimbabwe flies (Figure 1) and between the two fly 266 strains (Figure 5A).

267 Cosmopolitan and Zimbabwe female flies are expected to differ only with respect to odour 268 intensity, not quality - unless a blend of Z4-11Al and Z4-9Al produces a different aroma 269 than the Z4-11Al. This is indeed the case. A triangle test shows a very clear distinction 270 between Z4-11Al and a 3:10-blend of Z4-11Al and Z4-9Al (P=0.01; Figure 5C) mimicking 271 the scent of cosmopolitan and Zimbabwe flies, respectively.

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272 Discussion

273 Z4-11Al is a key ligand for the highly expressed OR10A6

274 Our olfactory perception of Z4-11Al, the female pheromone of the cosmopolitan strain of 275 D. melanogaster is highly sensitive and specific. We sense synthetic Z4-11Al at 10 ng/mL 276 (0.06 µmol/L) (Figure 4B) or at subnanogram amounts released by single flies (Figure 1; 277 Lebreton et al. 2017), and we distinguish between Z4-11Al and structurally similar 278 aldehydes, such as Z4-9Al and Z6-14Al (Figure 4B), and even a blend of Z4-11Al and Z4- 279 9Al (Figure 5C).

280 We here show that OR10A6 L287P (OR10A6), a very highly expressed human olfactory 281 receptor (Saraiva et al. 2019), is specifically tuned to Z4-11Al (Figure 3). Other human 282 ORs known to respond to odour-active aldehydes that are, for example, part of fruit 283 aromas (Nara et al. 2011, de March et al. 2015, Block 2018) are not nearly as responsive 284 to Z4-11Al as OR10A6 (Figure 3).

285 Remarkably, the panel also discriminated between Z4-11Al and a blend of Z4-11Al and 286 Z4-9Al, which explains how we discriminate cosmopolitan from Zimbawe flies (Figure 2, 287 5). Since OR10A6 is responsive to Z4-9Al (Figure 4), it is possible that modulation at the 288 OR level encodes this blend discrimination. Synergistic and antagonistic responses in 289 olfactory neurons to odour mixtures, compared with the individual components, show 290 that processing of odour interactions is not restricted to higher olfactory circuits, but 291 occurs even peripherally (Brann & Datta 2020, McClintock et al. 2020, Xu et al. 2020). 292 Broadly tuned, yet highly selective ORs are the basis for sensing a diverse odorant 293 environment with only a limited number of ORs (Geithe et al. 2017a, Block 2018, Saraiva 294 et al. 2019). Odorant interaction and encoding of odorant mixtures at the OR level very 295 substantially extends the receptive range of ORs.

296 It is further intriguing that we sense Z4-11Al released by single flies against the rich 297 bouquet emerging from a glass of wine (Figure 1A; Becher et al. 2018). Single ORs and 298 their key ligands play indeed a central role in olfactory object recognition, especially 299 against heterogenous backgrounds. Olfactory sensory neurons expressing high-affinity 300 ORs with low activation thresholds have been shown to become activated early during a 301 sniff and thus accentuate the response to behaviourally salient signals, while input from 302 other ORs is temporarily tuned down (Wilson et al. 2017, Arneodo et al. 2018, Bolding & 303 Franks 2018).

304 Taken together, our observations illustrate how a single OR contributes to olfactory 305 perception, in addition to combinatorial coding of odorant blends by arrays of several ORs 306 (Mainland et al. 2014). Sensitivity to odorants also depends on the number of olfactory 307 sensory neurons expressing the corresponding ORs (van der Linden et al. 2020). OR10A6

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308 is among the most highly expressed ORs in our nose (Verbeugt et al. 2014, Saraiva et al. 309 2019), which likely accounts for our high sensitivity to Z4-11Al.

310 Occurrence of Z4-11Al in human odour scenes

311 Highly abundant ORs are plausibly dedicated to odorants of critical physiological, 312 behavioral or ecological function, and this raises the question what Z4-11Al means to us. 313 Perception of the same odorant by insects and vertebrates is convergent, since the 314 respective ORs are built differently and lack a common phylogenetic root (Su et al. 2009, 315 Bear et al. 2016). It is, however, unclear whether shared perception is merely coincident, 316 or whether Z4-11Al is of mutual importance for flies and men.

317 What is the source of Z4-11Al in a human odorscape? Animals, plants and associated 318 microbes each release many hundreds of compounds and these volatile emissions change 319 with age, phenology and phyisological state (Knudsen et al. 1993, El-Sayed 2020, 320 Lemfack et al. 2018, Ljunggren et al. 2019). Z4-11Al has not been searched for, 321 synthetic standards are not commercially available and we can safely assume that the 322 occurrence of Z4-11Al is only incompletely known.

323 In food plants, Z4-11Al has been found in coriander and clementine (Chisholm et al. 324 2003, Eyres et al. 2005), and similar aldehydes are typical for fruit (e.g. Fischer et al. 325 2008, Chai et al. 2012). Monoenic aldehydes are perceived as "citrusy", but also as 326 "tallowy" since they contribute to the flavour of cooked or roast food and meat, including 327 rice, oils, fish, chicken and beef (Cha et al. 1992, Siegmund and Pfannhauser 1999, 328 Rochat and Chaintreau 2005, Roh et al. 2006, Yang et al. 2008, Oueslati et al. 2018), 329 and Z4-11Al has also been found in oxidized tallow (Shi et al. 2013).

330 The crested auklet, a sea bird, releases a tangerine-scented, social odour that signals 331 mate quality and contains (Z)-4-decenal as a main compound (Douglas et al. 2001, 332 Hagelin et al. 2003). Unsaturated aldehydes are part of human skin emanations (Duffy et 333 al. 2018) and serve as cues for detecting the presence of humans (Li et al. 2014). 334 Intriguingly, milk from humans and rabbits contains (E)-2-decenal and (Z)-2-undecenal, 335 respectively, and Z4-11Al has been found in rabbit anal glands, accelerating heartbeat 336 upon perception (Goodrich et al. 1978, Schaal et al. 2003, Duffy et al. 2018). Taken 337 together, Z4-11Al is found in human food, it might even be produced by ourselves and 338 could manifest food, social context, or both.

339 Emission of Z4-11Al by D. melanogaster

340 The vinegar fly is probably our first, involuntarily domesticated animal and it 341 accompanied our global expansion from out of Africa more than ten thousand years ago. 342 Cosmopolitan vinegar flies are associated with us on all continents and most climate 343 zones, they are strictly anthropophilic, depend on our dwellings and food for survival and

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344 they share our taste for fermenting food (Lachaise & Silvain 2004, Arguello et al. 2019). 345 D. melanogaster females, not males, produce dienic hydrocarbons that give rise to 346 monoenic aldehydes, which is why we smell the female flies (Everaerts et al. 2010, 347 Lebreton et al. 2017).

348 The sibling species D. simulans has also attained worldwide distribution in association 349 with humans, but is, unlike D. melanogaster, not a strict commensal and more rarely 350 found in households or buildings (Lachaise & Silvain 2004). D. simulans females do not 351 produce dienic hydrocarbons, which is a main element of the mating barrier between 352 these two sibling species. The cuticular hydrocarbon 7,11-HD promotes courtship in 353 cosmopolitan D. melanogaster, and suppresses interspecific matings with D. simulans, 354 owing to differential, species-specific coding of 7,11-HD in neural circuits mediating 355 reproductive behaviour (Billeter et al. 2009, Billeter & Wolfner 2018, Seeholzer et al. 356 2018, Sato and Yamamoto 2020).

357 Cosmopolitan and African D. melanogaster females also differ with respect to cuticular 358 hydrocarbons. The female-specific desaturase gene desat2, which affords 5,9-HD and Z4- 359 9Al, is functional only in African and not in cosmopolitan flies (Figure 2; Dallerac et al. 360 2000, Grillet et al. 2012). This hydrocarbon polymorphism yields a distinctive aldehyde 361 blend, which which is how we differentiate the scent of these two fly strains (Figure 5).

362 Species-specific differences in hydrocarbons align with corresponding aldehyde 363 signatures, that entail behavioural consequences in the flies. Z4-11Al attracts D. 364 melanogaster, but not males of the Zimbabwe strain, and has an antagonistic effect on 365 upwind flight attraction in D. simulans. This underlines the role of female-produced 366 volatile pheromones in long-range mate communication in Drosophila (Lebreton et al. 367 2017).

368 Chemosensory habitat adaptation and sensory drive

369 ORs are known to readily adapt to habitats and to dietary or social chemosensory niches, 370 in insects and vertebrates alike (Bear et al. 2016, Hughes et al. 2018, Saraiva et al. 371 2019). Splice forms of the fly receptor DmelOR69a are tuned to food odorants, including 372 yeast and citrus fruit flavours, and to the female pheromone, respectively (Lebreton et al. 373 2017). These splice forms provide an extra degree of freedom for acquisition of or 374 adaptation to new ligands, as long as they match the food and mate finding theme.

375 We are food and home to the flies, they depend on us and especially so in cold climate. If 376 Z4-11Al is part of human odour scenes, it provides a suitable signal for presence of 377 humans and related food resources, especially since it already participates in mate 378 communication in African D. melanogaster. Adaptation to a commensal lifestyle may have 379 provided a sensory drive and selection pressure towards a single pheromone, Z4-11Al.

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380 The interaction between natural and sexual selection has indeed ben shown to effect 381 cuticular hydrocarbon composition and mate recognition in D. melanogaster (Blows 382 2002). Habitat selection and specific mate recognition are tightly interconnected 383 (Paterson 1985, Endler 1992, Boughman 2002) and a chemosensory bias for an odorant 384 that is characteristic for human habitats may have driven a role of such an odorant in 385 sexual premating communication.

386 Conclusion

387 Sensing the scent of a single fly is out of the ordinary (Becher et al. 2018), and we even 388 differentiate between cosmopolitan and Zimbabwe female pheromones (Figure 5). But 389 only the discovery that a single, most highly expressed human OR (Figure 3; Saraiva et 390 al. 2019) is tuned to the fly pheromone Z4-11Al underlines the biological significance of 391 these observations.

392 Sensitive and specific perception suggests that Z4-11Al is found in human habitats, which 393 may have driven the role of Z4-11Al in mate communication in commensal D. 394 melanogaster flies. Convergent perception of Z4-11Al may stem from shared food 395 resources and associated microorganisms. This is reminiscent of dedicated olfactory 396 channels for geosmin that alert flies and humans about the presence of mould, which is 397 detrimental for all animals alike (Maga 1987, Stensmyr et al. 2012).

398 Ambient odorscapes contain countless chemicals of yet unknown activity. Our study 399 highlights how the identification of key OR ligands leads to the discovery of salient 400 messenger chemicals and delivers insights in how chemical communication interconnects 401 species across phylogenies. Regrettably, we can barely speculate what the fly pheromone 402 may mean to us and whether it signals food, social context, or both. Satisfying our 403 curiosity is an excellent reason to pursue, since the vinegar fly continues to afford 404 fundamental discoveries and studying fly sex perfumes may perhaps teach us about our 405 own.

406 Availability of data and materials

407 Data will be uploaded to Dryad.

408 Funding

409 This study was fincially supported by the Faculty of Landscape Architecture, Horticulture, 410 and Crop Production Science (SLU, Alnarp, Sweden).

Frey et al. - p. 13 (21)

411 Authors' contributions

412 PW, DK and PGB conceived the study. OR-screening by TF and DK, fly chemical analysis 413 by CAK, BB and MB. Sensory panel tests by EH, PGB and PW. EAW and EH synthesized 414 aldehydes. PW wrote text with input from all co-authors, all authors read and approved 415 the final manuscript version.

416 Acknowledgements

417 We thank the members of the sensory panel (Systembolaget, Stockholm) for evaluating 418 the fly scent.

419 Ethics approval and consent to participate

420 Not applicable.

421 Consent for publication

422 Not applicable.

423 Competing interests

424 Authors declare no competing interests.

425 Author details

426 1 Leibniz-Institut für Lebensmittel-Systembiologie an der Technischen Universität 427 München, Lise-Meitner Strasse 34, D-85354 Freising, Germany. 2 Department of Plant 428 Protection Biology, Swedish University of Agricultural Sciences, Box 102, 23053 Alnarp, 429 Sweden. 3 Department of Chemical Engineering, Mid Sweden University, Holmgatan 10, 430 85170 Sundsvall, Sweden. 4 Systembolaget AB, 103 84 Stockholm, Sweden.

431

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678 Legends

679 Figure 1.

680 Comparison of the olfactory resemblance of 10 ng synthetic Z4-11Al and the odour of D. 681 melanogaster female flies, of the cosmopolitan (A) and Zimbabwe strain (B), in water and

Frey et al. - p. 21 (21)

682 wine. Judges were asked whether or not the odours of two vials bear resemblance. Bars 683 marked with asterisks are significantly different (Students t-test).

684 Figure 2.

685 (A) The female pheromone of cosmopolitan D. melanogaster (Z)-4-undecenal (Z4-11Al) 686 and its precursor, (Z,Z)-7,11-heptacosadiene (7,11-HD) (Lebreton et al. 2017). Females 687 of the Zimbabwe strain produce in addition (Z,Z)-5,9-heptacosadiene (5,9-HD) and the 688 corresponding oxidation product is (Z)-9-nonenal (Z4-9Al). (B) Chromatograms of 689 headspace collections from cosmopolitan and Zimbabwe females, with peaks of Z4-9Al 690 and Z4-11Al highlighted. Zimbabwe flies produce Z4-9Al in a 2.6±0.7-fold amount 691 (n=10), compared to Z4-11Al.

692 Figure 3.

693 Screening of Z4-11Al (100 µmol/L) against 579 OR variants, including all 390 human OR 694 reference sequences (according to NCBI) and their most frequent variants, showed that

695 soleley OR10A6 L287P responded to Z4-11Al. Shown are means (n=2), mock control was 696 subtracted. Relative luminescence units (RLU), normalized to the response of OR1A1 to 697 (R)-(−)-carvone (30 µmol/L) (RLUmax).

698 Figure 4.

699 Dose response effect of synthetic Z4-11Al, Z4-9Al and Z6-11Al on OR10A6 L287P in HEK- 700 293 cells (A) and an odorant panel (B). Relative luminescence units (RLU) were mock 701 control-subtracted, and normalized to the highest Z4-11Al-induced signal of OR10A6

702 L287P (mean ± SD; n=3). For the panel test, compounds were formulated, from 0.01 ng 703 to 10 ng, in 10 mL redistilled water. For each dose, judges (n=9) evaluated a triangle of 704 two identical and one odd sample, they were asked to pick the odd sample. A significant 705 number of panelists distinguished Z4-11Al at 10 ng (Chi2- test; p<0.02).

706 Figure 5.

707 Olfactory discrimination of (A) cosmopolitan vs Zimbabwe D. melanogaster females, (B) 708 10 ng Z4-9Al vs 10 ng Z4-11Al, and (C) a 10:3-ng blend of Z4-9Al and Z4-11Al vs 10 ng 709 Z4-11Al. Judges were asked to point out the odd sample, among three vials containing 710 two identical samples. Bars marked with asterisks are significantly different (Chi2-test). 711 Vials containing synthetic compounds and fly odour were filled in 10 mL redistilled water.

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Cosmopolitan Zimbabwe Z4-11Al Z4-11Al bioRxiv preprint doi: https://doi.org/10.1101/2020.12.07.414714; this version posted December 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

O (Z,Z)-5,9-Heptacosadiene (5,9-HD) Z4-9Al

(Z,Z)-7,11-Heptacosadiene (7,11-HD) O

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Cosmopolitan Z4-11Al •

•

• Z4-9Al Zimbabwe Drosophila melanogaster bioRxiv preprint doi: https://doi.org/10.1101/2020.12.07.414714; this version posted December 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Human OR Screening with Z4-11Al - Cell-based Luminescence Assay 0.06 OR10A6 0.05 max

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RLU/RLU Z6-11Al bioRxiv preprint doi: https://doi.org/10.1101/2020.12.07.4147140.8 ; this version posted December 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.Z4-9Al All rights reserved. No reuse allowed without permission.

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1 10 28.1 32.9 47.8 100 µmol/L

Dose Response - Odor Panel B 100 * % Z4-11Al Z6-11Al Z4-9Al

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