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1 Mosquito indolergic receptors belong to an ancient and functionally conserved Dipteran gene 2 lineage 3 4 R. Jason Pitts1† 5 Shan Ju Shih1 6 Jonathan D. Bohbot2†* 7 8 9 10 1 Department of Biology, Baylor University, Waco, Texas, United States of America 11 2 Department of Entomology, The Hebrew University of Jerusalem, Rehovot, 76100, Israel 12 13 14 15 † R. Jason Pitts and Jonathan D. Bohbot contributed equally to this work 16 * Correspondence: [email protected] 17

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18 Abstract

19 Diptera is a megadiverse group of with sophisticated chemical detection systems, which

20 exploits an incredible variety of ecological niches. Among the vast array of odorants in natural

21 environments, indoles stand out as playing crucial roles in mediating behavior. In mosquitoes,

22 indolic compounds are detected by an ancient class of conserved indolergic Odorant Receptors

23 (indolORs). In this study, we have identified a set of 92 putative indolOR genes encoded in the

24 genomes of Nematoceran and Brachyceran flies, resolved their phylogenetic relationships, and

25 defined conserved elements in their gene structures. Further, we have quantified indolOR transcript

26 abundance in the antennae of the housefly, Musca domestica, and have characterized MdomOR30a

27 as a skatole receptor using a heterologous expression system. The presence of indolORs in

28 operating in different ecological contexts suggests that indoles act as pleiotropic signals for resource

29 exploitation at multiple developmental stages. Further characterization of indolORs will impact our

30 understanding of chemical ecology and will provide targets for the development of novel

31 odor-based tools that can be integrated into existing vector surveillance and control programs.

32

33 Introduction

34 detect, track and locate a variety of resources in rapidly changing environmental

35 conditions. Chemical cues are amongst the most predominant ecological drivers of behavior

36 and exert major influences on resource acquisition [1, 2]. Perhaps nowhere in animal lineages is this

37 dependency on olfactory information more evident than in , where accurate and timely

38 chemical detection is necessary for life history traits such as foraging, toxin and predator avoidance,

39 egg deposition, and mate selection. Indeed, insects, like other animals, possess highly evolved

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40 chemosensory systems that have allowed this large and diverse taxonomic group to exploit a

41 dazzling array of ecological niches [3–8].

42 Despite this tremendous diversity, some interesting convergent principles may underlie animal

43 olfactory-mediated behaviors. For example, highly ubiquitous compounds like indoles, which are

44 produced by plants and microorganisms, can either attract or repel insects [9] and can evoke either

45 pleasant or repugnant responses in humans [10, 11]. The behavioral valency for insects and the

46 perceptual quality for humans should depend on the quantity of indolic compounds emitted from

47 specific sources. While the molecular basis and ecological significance of indole sensing by humans

48 is unknown, the genomes of mosquitoes encode a small number of highly selective and exquisitely

49 sensitive indolergic Odorant Receptors (indolORs). IndolORs are remarkably conserved in

50 mosquitoes [12–14], considering the highly dynamic nature of this gene family [12] and the ancient

51 origin of the Anophelinae-Culicinae split that occurred 145–226 million years ago [15–17].

52 It is unclear whether indoles act as oviposition cues or as animal-host indicators [18, 19].

53 Furthermore, these heterocyclic compounds contribute to floral scent [20, 21] and are preferred by

54 A. aegypti in the context of plant-host attraction [22]. The complex developmental expression

55 patterns [12] and evolution of mosquito indolORs [13, 23, 24], in terrestrial adults and aquatic larvae,

56 suggest that these two compounds play key roles in regulating inter-specific interactions in addition

57 to their stage-specific functions. Mosquito species belonging to the subfamilies, Anophelinae and

58 Culicinae, express both an indole-selective (OR2) and a skatole-selective (OR10) receptor. In

59 Culicine mosquitoes, including members of the genera Aedes, a third paralog named OR9 operates

60 as a supersensitive skatole receptor during the aquatic stage [24]. The expression of indolORs in

61 aquatic larvae [12] and in terrestrial blood- and nectar-feeding adults of different sex and species

62 [23, 25] suggests that indolic compounds may have the intriguing property of influencing mosquito

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63 behaviors in multiple ecological contexts, including larval foraging, animal- and plant-host seeking,

64 and oviposition site selection [19].

65 This paradigm may have important implications for olfactory coding whereby mosquitoes and

66 other insects rely on various odor blends composed of combinations of commonly-occurring

67 chemical signals, such as indoles, plus resource-specific odorants. It is therefore of interest to seek

68 additional evidence in support of the proposition that other flies utilize parsimonious indole response

69 mechanisms to optimize olfactory-mediated resource acquisition. Such findings would inform our

70 understanding about the natural principles that have influenced the evolution of insect odor coding

71 and behavior.

72

73 Results

74 The dipteran indolOR gene family is phylogenetically ancient.

75 IndolORs are encoded by three genes in the yellow fever mosquito, Ae. aegypti (ORs 2, 9, 10).

76 We used the conceptual translations of these genes in homology-based searches to explore the

77 conservation of indolORs within the Diptera, focusing on species of medical and veterinary

78 relevance in the families Culicidae, Glossinidae, Muscidae, and . The primary amino

79 acid sequences of indolORs were used to build phylogenetic trees based on the Maximum-likelihood

80 method (Fig. 1). Our analysis reveals a clear distinction in the evolutionary histories of indolORs

81 between the lower flies () and higher flies (), which may reflect novelties in

82 their chemoreceptive functionalities (Fig. 1).

83 Each of the indolORs exist almost exclusively as single-copy genes within the genomes we

84 examined (Fig. 1). Subtle differences in the number of indolORs across genomes suggest single gene

85 losses or gains. For example, the Anophelines generally encode two indolOR homologs, ORs 2 and

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86 10, with species in the Cellia subgenus encoding a second OR10 paralog, which we have named

87 indolOR10b (Fig. 1). The Culicines encode three indolORs, including a distinct OR9 group (Fig. 1).

88 The genomes of Lutzomyia and Phlebotomus sandflies from the Psychodidae family, one of the

89 oldest families of Diptera, also encode two indolORs [30, 31]. Most of the Brachyceran species

90 encode exactly three candidate homolog indolORs, including single representative genes for OR30a,

91 OR43a, and OR49b. We identified two of three homologs in H. irritans, with OR49 apparently

92 absent from the genome. Interestingly, we identified an additional paralog of OR30a in the genomes

93 of S. calcitrans and H. irritans, which we have named OR30b, plus two additional paralogs in M.

94 domestica, named OR30b and OR30c (Fig. 1). OR30a and OR49b clades are completely resolved

95 with respect to the nematoceran lineages. Their branching pattern suggests that these genes evolved

96 more recently than the OR43a clade as indicated by a potential common ancestor with the

97 phylogenetically ancient Psychodidae OR2 and OR3 genes. Indeed, the branch leading to the

98 Brachyceran OR43a clade indicates substantial amino-acid divergence suggesting a common

99 ancestor with sandflies.

100

101 Dipteran indolOR homologs are highly divergent.

102 We examined the degree of homology across Diptera by aligning the amino acids sequences of

103 a selected subset of receptors from A. aegypti, P. papatasi, L. longipalpis, D. melanogaster, and M.

104 domestica (Fig. 2A). Amino acid conservation increases from the N- to C-terminus across all

105 indolORs and is highest at the extreme C-terminal end of the receptors, encoded by their final exons.

106 This C-terminal conservation is a general feature of insect odorant receptors [14]. Overall amino

107 acid identities are typically between 30-40%, even when comparing different indolORs and across

108 lower and higher flies (Fig. 2B). Highest identities of 50-80% are observed for intraspecies indolORs

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109 and for some intrafamily orthologs. For example, A. aegypti OR9 and OR10 are 70% identical, while

110 P. paptasi OR3 and L.longipalpis OR3 are 81% identical (Fig. 2B). Another illustration of

111 conservation that likely reflects their evolutionary histories is the relationships between the M.

112 domestica and D. melanogaster indolORs. The OR30a, OR43a, and OR49b orthologs from these

113 two species share 58%, 45%, and 63% amino acid identities, respectively (Fig. 2B). However,

114 MdomOR30a shares only 31% amino acid identity with DmelOR43a, but 46% amino acid identity

115 with DmelOR49b, supporting a more distant evolutionary relationship between the OR30a and

116 OR43a clades.

117

118 Gene structure and microsynteny of Dipteran indolORs is conserved.

119 Patterns of intron conservation (Fig. 3) and chromosomal synteny (Fig. 4) further support their

120 evolutionary relationships. For example, the preservation of ancestral introns (A1-A6) indicate a

121 common gene lineage for indolORs, while nematoceran-specific introns (N1, N2) indicate divergent

122 evolution between lower and higher flies (Fig. 3). A2 is absent from the Culicidae lineage. Clustering

123 of the OR2, 9, and10 genes, as well as conservation of neighboring orthologs in Culicidae indicated

124 preservation of chromosomal segments in nematoceran (Fig. 4). Although the brachyceran indolORs

125 30a, 43a, and 49b are dispersed throughout their respective genomes, examples of preservation of

126 orthologs are readily identified, especially for indolOR49b across the Drosophilidae, Muscidae, and

127 Glossinidae families (Fig. 4).

128

129 Musca domestica indolORs are expressed in the adult antennae.

130 To examine the functional expression of brachyceran indolORs, we chose to focus on Musca

131 domestica, a major global pest species that serves as a mechanical vector for bacterial pathogens,

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132 especially species in the genus, Shigella, that cause dysentery [32, 33]. We conducted an RNA

133 sequencing analysis of antennae to assess transcript abundance as proxy for receptor expression.

134 IndolORs were detected at levels suggesting that they are functional in this appendage in both sexes

135 (Fig. 5). Abundances for all other transcripts, including additional chemoreceptors, are provided in

136 Supplemental data. Among the antennal-expressed indolORs in M. domestica, OR30a displayed the

137 highest abundance with an average TPM of 18.2 across both sexes, followed closely by OR43a with

138 an average TPM of 17.7 (Fig. 5). The abundances of OR30b and 49b were about 3 to 5-fold lower,

139 which could reflect reduced expression in individual ORNs, the cumulative number of ORNs

140 expressing each receptor, or both.

141

142 MdomOR43a is a skatole receptor.

143 To test the potential functional orthology with mosquito indolORs, we utilized Xenopus laevis

144 oocytes as a heterologous expression platform and the two-electrode voltage clamping technique to

145 record the responses of MdomOR30a to a panel of structurally related indolic compounds. When

146 co-expressed with MdomORco, MdomOR30a was highly selective towards skatole, followed by

147 2,3-dimethylindole and indole with a kurtosis value of 7.05 (Fig. 6A). MdomOR30a also displayed

148 concentration-dependency of indolic responses over several orders of magnitude (Fig. 6B). With a

149 half maximal effective concentration (EC50) in the low-mid nanomolar range, skatole was the most

150 potent agonist odorant and was an order of magnitude lower than sensitivities to indole or 2,3-

151 dimethylindole (Fig. 6C). This high degree of sensitivity to skatole is similar to those that have been

152 reported for nematoceran, specifically mosquito, indolORs (Table 1).

153 Table 1. List of cognate pheromone and kairomone receptors deorphanized in the Xenopus laevis

154 expression system.

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IndolOR Species Ligand EC50 (nM) Reference

OR2 Ae. aegypti Indole 370 23

Cx. quinquefasciatus Indole 280 34

Tx. amboinensis Indole 167 24

An. gambiae Indole 88 35

OR9 Ae. aegypti 3-Methylindole 4.8 13

OR10 Ae. aegypti 3-Methylindole 109 23

Cx. quinquefasciatus 3-Methylindole 90 36

Tx. amboinensis 3-Methylindole 870 24

M. domestica 3-Methylindole 331 this study (Fig. 6C)

155

156 Discussion

157 In this study, we propose that the brachyceran OR30a is an indolOR by virtue of its ligand

158 selectivity, nanomolar sensitivity towards 3-methylindole, and gene structure. Until now, IndolORs

159 were considered to be a Culicidae-specific expansion due to their ligand-binding characteristics [13,

160 24, 34–40] and amino-acid sequence conservation (50-70%) [12]. Candidate indolORs outside the

161 Culicidae family display an amino-acid sequence identity significantly lower than within

162 mosquitoes. Relaxing this qualifying criterion allowed us to identify putative indolOR homologs

163 within the phylogenetically ancient Psychodidae family and more significantly in more distantly-

164 related flies. OR30a, OR43a and OR49b form distinct and brachyceran-specific clades as reflected

165 by their low amino-acid conservation (34-43%) with their mosquito counterparts and diverse

166 syntenic relationships (Figs. 2, 4)

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167 IndolORs are expressed in fly species that exhibit a great diversity of diets, oviposition habitats,

168 and chemical ecologies. For example, mosquitoes, sand flies, stable flies, horn flies and tsetse flies

169 are all hematophagous as adults. In the mosquitoes and sandflies, only the females bloodfeed while

170 in the latter three groups, both sexes bloodfeed. Some of the brachyceran flies studied here, with the

171 exceptions of D. melanogaster and Glossina species, produce maggots that feed on decaying matter

172 (e.g., manure). The larvae of tsetse flies spend nearly all of their larval development within the

173 female uterus, feeding on specialized glands.

174 Drosophila melanogaster also has 3 three putative indolORs, whose function are currently

175 unknown. While OR43a and OR49b are expressed in the adult antennae [41], OR30a is only

176 expressed during the larval stage [41–43], suggesting distinct developmental roles for indole

177 detection. Little is known about the role of indoles in the chemical ecology of these insects. However,

178 indole and 3-methylindole have been identified as potential food and oviposition attractants [44–

179 46]. Expanding our analysis to additional dipteran genomes will likely uncover additional, divergent

180 indolORs, thereby providing an opportunity to explore the roles of indoles in these species.

181 Female M. domestica flies are attracted by fetid scents signaling suitable oviposition sites, which

182 are mainly provided by animal manure [47]. Similar odors are released by Sapromyiophilous plants

183 to attract flies for pollination purposes [20]. In both cases, the attraction of M. domestica to

184 oviposition and feeding sites is in part mediated by indoles (indole and 3-methylindole) sensitive

185 antennal receptors in females as well as in males [47, 48] (Fig. 7). The discovery of a 3-methylindole

186 sensitive and selective OR30a that is expressed in the antennae of the housefly, Musca domestica,

187 provides a candidate molecular mechanism for the observed attraction of this insect to 3-

188 methylindole [45, 49, 50]. Further experiments, such as gene knockout, will be required to establish

189 a causal link between OR30a and 3-methylindole-mediated olfactory behaviors in flies. In addition,

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190 cellular-level expression studies and single-sensillum recordings are needed to resolve global

191 antennal patterns of sensillar expression and the relationships between indolOR-expressing OSNs

192 and their physiological functions in peripheral odor signaling.

193

194 Conclusions

195 In this study, we provide genetic, evolutionary and functional evidence supporting the hypothesis

196 that M. domestica OR30a is a selective indolOR. The reconstitution of a sensitive and selective 3-

197 methylindole receptor, recapitulating previous physiological studies [47, 48], paves the way for

198 exploring the roles of indolORs to indole-sensing in insects exhibiting a broad variety of feeding

199 and oviposition habits. Finally, the identification of indolORs across multiple dipteran lineages

200 illustrates the multiple roles of indoles in insect chemical ecology and underscores their importance

201 in olfactory coding in a diverse group of terrestrial animals.

202

203 Methods

204 List of Species

205 Nematocera

206 Culicidae:

207 Aedes aegypti (L.) Aedes albopictus (Skuse), Anopehels albimanus (Wiedemann),

208 Anopheles albimanus (Wiedemann), Anopheles arabiensis (Patton), Anopheles atroparvus

209 (Van Thiel), Anopheles bwambae (White), Anopheles christyi (Newstead & Carter),

210 Anopheles coluzzii (Coetzee & Wikerson), Anopheles culicifacies (Giles), Anopheles

211 darlingi (Root), Anopheles dirus (Peyton & Harrison), Anopheles epiroticus (Linton &

212 Harbach), Anopheles farauti (Laveran), Anopheles funestus (Giles), Anopheles gambiae

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213 (Giles), Anopheles maculatus (Theobald), Anopheles melas (Theobald), Anopheles merus

214 (Dönitz), Anopheles minimus (Theobald), Anopheles quadriannulatus (Theobald),

215 Anopheles sinensis (Wiedemann), Anopheles stephensi (Liston), Culex pipiens

216 quinquefasicatus (L.)

217 Psychodidae: Phlebotomus papatasi (Scopoli), Lutzomyia longipalpis (Lutz & Neiva)

218 Brachycera

219 Muscidae: Musca domestica (L.), Stomoxys calcitrans (L.), Haematobia irritans (L.)

220 Glossinidae: Glossina austeni (Newstead), Glossina brevipalpis (Newstead), Glossina

221 fuscipes (Newstead), Glossina morsitans (Westwood), Glossina pallidipes (Austen),

222 Glossina palpalis (Robineau-Desvoidy)

223 Drosophilidae: Drosophila melanogaster (Meigen)

224

225 Identification of IndolORs and Gene Annotations

226 IndolOR homologs were identified in the genomes if Dipteran species listed above via tBLASTn

227 or BLASTp searches against available genome assemblies on the National Center for Biotechnology

228 Information (www.ncbi.nlm.nih.gov) or Vectorbase (www.vectorbase.org). Gene annotations were

229 corrected based upon multiple amino acid alignments using Geneious Prime2019 software

230 (Biomatters Limited, USA), as well as conservation of intron positions. Chromosomal synteny was

231 determined by identifying conserved orthologs encoded in either the 5’ or 3’ directions of indolORs

232 on their respective genome assemblies.

233

234 Phylogenetic Analysis

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235 Conceptual amino acid sequences of 92 indolORs were aligned using MAFFT version 7 [26]. The

236 evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based

237 model [27]. The tree with the highest log likelihood (-20016.97) is shown. The percentage of trees

238 in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the

239 heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a

240 matrix of pairwise distances estimated using the JTT model, and then selecting the topology with

241 superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number

242 of substitutions per site. This analysis involved 92 amino acid sequences with a total of 431 positions

243 in the final dataset. Evolutionary analyses were conducted in MEGA X [28, 29]. One thousand

244 bootstrap pseudoreplicates were performed and best tree was obtained, where branches with >50%

245 bootstrap were retained.

246

247 Chemical reagents

248 The chemicals used for the deorphanization of MdomOR30a were obtained from Acros Organics

249 (Morris, NJ), Alfa Aesar (Ward Hill, MA), and Thermo Fisher Scientific (Waltham, MA) at the

250 highest purity available. Compounds were initially dissolved to 1M in 100% DMSO to generate

251 stock solutions and serially diluted in ND96 perfusion buffer (96mM NaCl, 2mM KCl, 5mM MgCl2,

252 0.8mM CaCl2, and 5mM HEPES) for oocyte recordings.

253

254 Two-electrode voltage clamp of Xenopus laevis oocytes

255 MdomOrco and MdomOR30a coding regions were obtained by de novo synthesis into a pENTR

256 plasmid from a commercial source (Twist Bioscience, South San Francisco, CA). Subcloning into

257 the pSP64t expression plasmid was accomplished using the Gateway® LR clonase II® enzyme (Life

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258 Technologies, Carlsbad, CA). Individual cRNAs were synthesized in vitro from linearized pSP64t

259 using the mMESSAGE mMACHINE® SP6 kit (Life Technologies). Stage V-VII Xenopus laevis

260 oocytes were obtained from a commercial source (Xenopus1, Dexter, MI) and incubated in ND96

261 incubation media (96 mM NaCl, 2mM KCl, 5mM HEPES, 1.8mM CaCl2, 1mM MgCl2, pH 7.6)

262 supplemented with 5% dialyzed horse serum, 50 µg/mL tetracycline, 100µg/mL streptomycin,

263 100µg/mL penicillin, and 550 µg/mL sodium pyruvate. Oocytes were injected with 27.6 nL (27.6 ng

264 of each cRNA) of RNA using a Nanoliter 2010 injector (World Precision Instruments, Inc., Sarasota,

265 FL). Odorant-induced currents of oocytes expressing AgamOrco and MdomOR30a were recorded

266 using the two-microelectrode voltage-clamp technique (TEVC) using an OC-725C oocyte clamp

267 (Warner Instruments, LLC, Hamden, CT), while maintaining a holding potential of -80 mV. Data

268 acquisition and analysis were carried out with the Digidata 1550B digitizer and pCLAMP10

269 software (Molecular Devices, Sunnyvale, CA, USA). A panel of 12 structurally-related indolic

270 compounds: Indole-3-acetic acid, 2,3-dioxoindoline, 4-hydroxyindole, quinoline, indole-3-

271 carboxyaldehyde, 2,3-dimethylindole, 3-methylindole, indole, methyl indole-3-carboxylate, 2-

272 methylindole, 2-oxindole, and methyl salicylate, were used to determine the most efficacious ligand

273 for MdomOR30a (n=14 oocytes). Compounds were perfused at 10-4 M for 10 s and current was

274 allowed to return to baseline between administrations. Kurtosis was calculated for the raw

275 amplitudes and the KURT function in Microsoft Excel 365. Concentration-dependent responses

276 were determined by perfusing oocytes with indole, 3-methylindole, or 2,3-dimethylindole across a

277 log-molar range of 10-9 to 10-3 M. Data analyses were performed using GraphPad Prism 8 (GraphPad

278 Software Inc., La Jolla, CA, USA). Compounds were perfused for up to 30s or until peak amplitude

279 was reached. Current was allowed to return to baseline between chemical compound administrations.

280

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281 Supplementary information

282 Supplementary information accompanies this paper at:

283 Additional file1: Tables S1–S4. Table S1: List of indolORs, abbreviations, and amino acid

284 sequences by species. Table S2: Raw oocyte recording data. Table S3: IndolOR gene annotations.

285 Table S4: Nematocera and brachycera syntenic relationships.

286

287 Acknowledgements

288 We acknowledge Robert Huff (Baylor University) for assistance with electrophysiology and Amir

289 Dekel (Hebrew University of Jerusalem) for assistance with kurtosis calculation.

290

291 Authors’ contributions

292 RJP and JDB conceived and designed the study. RJP and JDB conducted the bioinformatics

293 analyses. SJS performed electrophysiology experiments. RJP and JDB analyzed the data and wrote

294 the manuscript. All authors read and approved the final manuscript.

295

296 Funding

297 This work was supported by Baylor University (RJP) and by a grant from the Israel Science

298 Foundation (1990/16).

299

300 Availability of data and materials

301 The datasets supporting the conclusions of this article are included within the article and its

302 additional files.

303

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304 Ethics approval and consent to participate

305 Not applicable.

306

307 Competing interests

308 The authors declare that they have no competing interests.

309

310 Author details

311 1 Department of Biology, Baylor University, Waco, Texas, United States of America 312 2 Department of Entomology, The Hebrew University of Jerusalem, Rehovot, 76100, Israel 313

314

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315 316 Figure Legends 317 318 Figure 1. Dipteran indole-sensitive odorant receptor proteins (IndolORs) are phylogenetically 319 ancient. Maximum Likelihood analysis of 92 fly indolORs homologs (Bootstrap support from 1000 320 replications color coded as indicated by the legend). Nematocera and Brachycera indolOR subclades 321 are indicated by vertical grey and black bars, respectively. The bootstrap value supporting the 322 Psychodidae and OR43a clades is shown above the split. Scale bar represents 0.2 changes per 323 residue. For a full listing of indolORs and abbreviations, see Table S1. 324 325 Figure 2. Dipteran indole-sensitive odorant receptors (IndolORs) proteins are highly 326 divergent. A) Amino-acid sequence identity (grey) of indolORs from Aedes aegypti (Aaeg), 327 Lutzomyia longipalpis (Llon), Phlebotomus papatasi (Ppap), Drosophila melanogaster (Dmel) and 328 Musca domestica (Mdom) indolORs (OR2, OR9, OR10, OR3, OR43a, OR30a & OR49b). 329 Percentage of amino-acid sequence identity is shown below. B) Amino-acid sequence identity 330 matrix. Intensity of shading indicates magnitudes of homologies. 331 332 Figure 3. Dipteran indole-sensitive odorant receptor genes (IndolORs) exhibit conserved 333 intron-exon structure. Exons (e1-8), positions and phases of ancestral (A1-6) and nematoceran- 334 specific (N1, N2) introns in fly indolOR genes. 335 336 Figure 4. Microsyntenic relationships of dipteran indole-sensitive odorant receptor genes 337 (IndolORs). Relative positions of IndolORs (red) and conserved neighboring genes (each homolog 338 groups are color-coded) on supercontigs (SC), scaffold (S) and chromosomes. Direction of 339 transcription is indicated by arrows. The color coding only applies within OR groups. Distances 340 between genes are not drawn to scale. For a full listing of genes and abbreviations, see Supplemental 341 Table 2. 342 343 Figure 5. M. domestica IndolORs are expressed in adult male and female antennae. A) IndolOR 344 transcripts, 30a, 30b, 43a, and 49b, and Orco are expressed in the antennae of male and female 345 housefly. Numbers represent Transcripts Per Million (TPM). 346 347 Figure 6. M. domestica OR30a is a skatole receptor. A) MdomOR30a is a 3-methylindole 348 (skatole) selective receptor. Mean normalized oocyte responses using a panel of indolic compounds 349 (n=10). B) Representative current traces elicited by 3-methylindole, indole and 2,3 dimethylindole. 350 Arrowheads above traces indicate stimulus onset, while numbers indicate log molar concentrations 351 of ligands. C) MdomOR30a responds to 3-methylindole in the nanomolar range (EC50 = 331 nM; n 352 = 7). Both indole (EC50 = 5,196 nM; n = 10) and 2,3 dimethylindole (EC50 = 7,040 nM; n = 7) are 353 less potent ligands, activating MdomOR30a in the high-nanomolar to low-micromolar range. 354 355 Figure 7. The MdomOR30a-3methylindole cognate pair mediates oviposition selection and 356 food seeking behaviors. The expression of MdomOR30a in male and female houseflies supports a 357 dual role of 3-methylindole in the attraction of flies to oviposition sites and food sources. 358 359 360

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20 LlonOr2 Psychodidae bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.236091; this version posted PpapOr2 August 4, 2020. The copyright holder for this preprint LlonOr3 (which was not 48certified by peer review) is the author/funder, who has granted bioRxiv a licensein tod olORdisplay thes preprint in perpetuity. It is made available under aCC-BY-NC-ND PpapOr3 4.0 International license. DmelOr43a MdomOr43a HirrOr43a ScalOr43a GbreOr43a GfusOr43a OR43a GpalpOr43a GausOr43a Bootstrap Support GmorOr43a >90% GpallOr43a >80% AaegOr2 >70% AalboOr2 >60% CquiOr121 >50% AmerOr2 AquaOr2 AcolOr2 0.20 AaraOr2 AgamOr2 AbwaOr2 AmelOr2 OR2 AsteOr2 N AmacOr2 AfunOr2 AminOr2 AculOr2 AepiOr2 AchrOr2 AfarOr2 AdirOr2 AsinOr2 AatrOr2 AdarOr2 AalbiOr2 CH CquiOr120 3 AalboOr9 OR9 AaegOr9 N CquiOr21 AalboOr10 AaegOr10 AminOr10b AculOr10b AfunOr10b AsteOr10b AfarOr10b AdirOr10b AsinOr10 AatrOr10 AdarOr10 CH AalbiOr10 3 AminOr10a OR10 AculOr10a N AfunOr10a AsteOr10a AmacOr10 AfarOr10a AdirOr10a AepiOr10 AchrOr10 AaraOr10 AmelOr10 AgamOr10 AcolOr10 AbwaOr10 AmerOr10 AquaOr10 DmelOr49b MdomOr49b ScalOr49b GausOr49b GmorOr49b GpallOr49b OR49b GfusOr49b GpalpOr49b GbreOr49b DmelOr30a MdomOr30a HirrOr30a ScalOr30a MdomOr30b ScalOr30b HirrOr30b GbreOr30a OR30a GfusOr30a GpalpOr30a GausOr30a GpallOr30a GmorOr30a MdomOr30c A bioRxivAaegOR2 preprint doi: https://doi.org/10.1101/2020.08.04.236091; this version posted August 4, 2020. The copyright holder for this preprint (whichAaegOR10 was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made AaegOR9 available under aCC-BY-NC-ND 4.0 International license. LlonOR2 PpapOR2 LlonOR3 PpapOR3 DmelOR43a DmelOR30a DmelOR49b MdomOR43a MdomOR30a MdomOR30b MdomOR49b

100 % 0 1 50 100 150 200 250 300 350 377

Aaeg Aaeg Llon Ppap Llon Ppap Dmel Dmel Dmel Mdom Mdom Mdom Mdom B OR9 OR10 OR2 OR2 OR3 OR3 OR43a OR30a OR49b OR43a OR30a OR30b OR49b AaegOR2 51 53 37 35 39 38 34 37 37 33 41 38 40 AaegOR9 70 35 37 38 37 36 37 37 33 43 39 40 AaegOR10 39 37 39 38 36 38 35 35 43 40 42 LlonOR2 67 55 54 30 29 32 31 33 31 33 PpapOR2 54 54 32 30 31 31 34 33 34 LlonOR3 81 31 33 31 32 35 33 35 PpapOR3 31 32 32 32 37 34 35 DmelOR43a 27 30 45 31 29 32 DmelOR30a 44 32 58 50 44 DmelOR49b 27 46 39 63 MdomOR43a 32 30 30 MdomOR30a 64 52 MdomOR30b 44 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.236091; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Intron N1 A1 A2 N2 A3 A4 A5 A6 Phase 00 1 0 2 0 0 0 Exon e1 e2 e3 e4 e5 e6 e7 e8

1 50 100 150 200 250 300 350 390 OR2 Aaeg & AlbOR2 CquiOR121 Anopheles spp. OR2

OR9 Aaeg & AalbOR9 CquiOR120

OR10 Aaeg & AalbOR10 CquiOR21

Anopheles spp. OR10 AdarOR10 AmacOR10

Sandflies OR2 & OR3 Ppap & LlonOR2

Ppap & LlonOR3

OR43a DmelOR43a

Mdom & ScalOr43a

HirrOr43a

Glossina spp. OR43a

GpalpOR43a

OR49b DmelOR49b MdomOr49b

ScalOr49b

Glossina spp. OR49b

OROR30a DmelOR30a

Mdom & ScalOr30a/b

Glossina spp. OR30a

MdomOr30c

HirrOr30a

HirrOr30b bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.236091; this version posted August 4, 2020. The copyright holder for this preprint OR2,(which OR9 was not& OR10certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. AAEL007703 AAEL022793 AAEL006002 AAEL006010 AAEL007696 AAEL020317 AAEL025006OR9 OR2 OR10 AAEL006004 AAEL020457 AAEL006001 A. aegypti 2

CPIJ014387 OR120 OR121 OR120 CPIJ014389 CPIJ014388 CPIJ014385 C. quinquefasciatus SC 3.528

AGAP028568 AGAP029527 AGAP009523 AGAP009524 AGAP009515 AGAP028599 AGAP009518 OR2 OR10 AGAP009521 AGAP009536 AGAP009525 A. gambiae 3R

ASTE011377 ASTE011375 ASTE011367 ASTE011378 ASTE011376 ASTE011371 OR2 OR10 ASTE011368 ASTE011352 ASTE011366 A. stephensi SC KB664644

OR43a OR43a MDOA003658 M. domestica S 172

OR43a SCAU007891 S. calcitrans SC KQ080208

OR43a FBgn0026602 D. melanogaster 2R

OR49b MDOA008225 MDOA002637 MDOA008845 MDOA012882 OR49bMDOA002363 MDOA014884 MDOA010617 MDOA009422 MDOA011716 M. domestica S 18733

SCAU008091 SCAU016500 SCAU014301 OR49bSCAU002452 SCAU014957 SCAU015467 SCAU011380 S. calcitrans SC KQ080114

FBgn0034063 FBgn0034065 FBgn0034067 FBgn0034069 OR49b FBgn0010590 FBgn0034066 FBgn0034068 FBgn0034070 FBgn0050421 D. melanogaster 2R

GAUT005600 GAUT005585 GAUT005583 GAUT005584 GAUT005572 GAUT005610 GAUT005586 GAUT005587 GAUT005588 GAUT005573 G. austeni S 121

GPAI004553 GPAI004552 GPAI004554 GPAI004537 GPAI004539 GPAI004557 GPAI004556 GPAI004555 GPAI004540 GPAI004538 G. pallidipes S 10

OR30a MDOA007519 MDOA002072 MDOA012481 OR30a MDOA001238 MDOA009614 MDOA015122 MDOA006573 M. domestica S 18939

FBgn0031753 OR30a FBgn0023090 FBgn0032945 FBgn0032949 FBgn0031752 FBgn0041245 FBgn0032946 D. melanogaster 2L bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.236091; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Orco 1318.5 1344.0 30a 20.5 15.9 30b 7.0 8.0 43a 16.1 19.3 49b 1.73 3.58 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.236091; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A available under aCC-BY-NC-ND 4.0 International license. 100 MdomOR30a-Orco

80

60

40

20

0 O OH O O Normalized (%) Normalized response OH CH3 CH3 O OCH3 O O O CH3 CH3 O N N N N N N N N N N N OH

Indole Quinoline 2-Oxindole

3-Methylindole 2-Methylindole Methyl salicilate 2,3-Dioxoindoline4-Hydroxyindole 2,3-Dimethylindole Indole-3-acetic acid

Indole-3-carboxyaldehyde Methyl indole-3-carboxylate

B C 3-Methylindole 1.0 3-Methylindole -9-8 -7.3-7 -6.3 -6 -5.3 Indole 2,3-Dimethylindole

0.8 400 nA 4 min Indole -8 -7 -6.3-6 -5.3 -5 -4 0.6

331 nM 5,196 nM 7,040 nM 50

400 nA 0.4 4 min 2,3 Dimethylindole -8 -7 -6.3-6 -5.3 -5 -4 0.2 Normalized Current Response (%)

0.0 nM μM 400 nA -9 -8 -7 -6 -5 -4 2 min Odorant (Log [M]) bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.236091; this version posted August 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

3-Methylindole Oviposition site

N

Feeding OR30a