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. 2017 Mar 3;42(4):333–341. doi: 10.1093/chemse/bjx011

Functional and Nonfunctional Forms of CquiOR91, an Odorant Selectivity Subunit of Culex quinquefasciatus

David T Hughes 1,3, Julien Pelletier 2,4, Suhaila Rahman 1, Sisi Chen 1,5, Walter S Leal 2, Charles W Luetje 1,✉
PMCID: PMC5964367  PMID: 28334229

Abstract

In Culex quinquefasciatus, CquiOR91 is the ortholog of 2 larvae-specific odorant receptors (ORs) from Anopheles gambiae (Agam\Or40, previously shown to respond to several odorant ligands including the broad-spectrum repellent N,N-diethyl-3-methylbenzamide, DEET) and Aedes aegypti (Aaeg\Or40). When we cloned full-length CquiOR91 from a Culex quinquefasciatus larval head RNA sample, we found 2 alleles of this OR, differing at 9 residues. Functional analysis using the Xenopus oocyte expression system and 2-electrode voltage clamp electrophysiology revealed one allele (CquiOR91.1) to be nonfunctional, whereas the other allele (CquiOR91.2) was functional. Receptors formed by CquiOR91.2 and Cqui\Orco responded to (−)-fenchone, (+)-fenchone, and DEET, similar to what has been reported for Agam\Or40. We also identified 5 novel odorant ligands for the CquiOR91.2 + Cqui\Orco receptor: 2-isobutylthiazole, veratrole, eucalyptol, d-camphor, and safranal, with safranal being the most potent. To explore possible reasons for the lack of function for CquiOR91.1, we generated a series of mutant CquiOR91.2 subunits, in which the residue at each of the 9 polymorphic residue positions was changed from what occurs in CquiOR91.2 to what occurs in CquiOR91.1. Eight of the 9 mutant versions of CquiOR91.2 formed functional receptors, responding to (−)-fenchone. Only the CquiOR91.2 Y183C mutant was nonfunctional. The reverse mutation (C183Y) conferred function on CquiOR91.1 , which became responsive to (−)-fenchone and safranal. These results indicate that the “defect” in CquiOR91.1 that prevents function is the cysteine at position 183.

Keywords: electrophysiology, insect, olfaction, polymorphism, receptor, Xenopus oocytes

Introduction

Olfaction is a critical driver of many insect behaviors, such as feeding, mating, and oviposition. Disease vector mosquitoes are no exception, using a variety of olfactory cues, as well as heat and visual cues, to locate and feed on humans (Gibson and Torr 1999). Olfactory cues are detected by multiple receptors, including odorant receptors (ORs) and members of the gustatory receptor (GR) family, as well as the ionotropic glutamate receptor-like IRs (Hallem et al. 2004; Lu et al. 2007; Benton et al. 2009; Carey et al. 2010; Wang et al. 2010; Erdelyan et al. 2012; DeGennaro et al. 2013; McMeniman et al. 2014). Insect ORs are a novel class of ligand (odorant) gated ion channel (Sato et al. 2008; Wicher et al. 2008) located on the dendrites of olfactory sensory neurons. These receptors are formed by an invariant subunit (the OR coreceptor subunit, known as Orco, Vosshall and Hansson 2011) that is highly conserved across species and one of a large number of highly variable odorant selectivity subunits (Krieger et al. 2003; Hallem and Carlson 2004; Larsson et al. 2004; Pitts et al. 2004; Jones et al. 2005; Nakagawa et al. 2005; Neuhaus et al. 2005; Benton et al. 2006; Vosshall and Stocker 2007). Both Orco and the odorant selectivity subunits are thought to contribute to the structure of the ion pore (Sato et al. 2008; Nichols et al. 2011; Pask et al. 2011).

The southern house mosquito, Culex quinquefasciatus, deploys a large repertoire of ORs, formed by Orco and 176 odorant selectivity subunits (Leal et al. 2013). Female Culex mosquitoes feed on birds and humans, thus acquiring and transmitting West Nile virus in the United States. Culex mosquitoes are also prominent vectors for other human diseases in the rest of the world, including filariasis and various types of encephalitis (Nasci and Miller 1996). Recent evidence suggests that C. quinquefasciatus is also a potential vector for Zika virus (Franca et al. 2016). Attempts to deorphanize OR genes from C. quinquefasciatus (Hughes et al. 2010; Pelletier et al. 2010; Xu et al. 2013) have been challenging in part because of the high degree of polymorphism. Indeed, Culex mosquitoes have the highest density of single-nucleotide polymorphism of mosquito species studied to date (Lee et al. 2012).

Comparative analyses have revealed that mosquito OR repertoires are highly diversified, with many species-specific expanded OR lineages and only a few orthologs conserved across species (Bohbot et al. 2007; Sánchez-Gracia et al. 2009; Pelletier et al. 2010). At the larval stage, mosquitoes express only a subset of ORs, including a conserved subgroup comprised of Agam\Or40 (AGAP002558), Aaeg\Or40 (AAEL005767), and CquiOR91 (CPIJ009579) (Bohbot et al. 2007; Xia et al. 2008; Leal et al. 2013). AgamOr40 was shown to respond to several compounds, including fenchone and the broad-spectrum repellent N,N-diethyl-3-methylbenzamide (DEET) (Xia et al. 2008). The relatively high conservation across orthologs at the protein level (45–56% identity), combined with the larvae-specific expression suggests that these ORs might contribute to mediating important larvae-specific behaviors in different mosquito species. Our attempts to clone CquiOR91 from larvae antennal tissues revealed the presence of 2 alleles, differing at 9 amino acid positions. This intriguing discovery prompted us to investigate the functional significance of these differences by examining the 2 different alleles of CquiOR91 in the Xenopus oocyte expression system.

Materials and methods

Materials

Xenopus laevis frogs were purchased from Nasco. The care and use of frogs in this study were approved by the University of Miami Animal Research Committee and meet the guidelines of the National Institutes of Health. Odorants and other chemicals were from Sigma–Aldrich.

Mosquitoes

Culex quinquefasciatus mosquitoes used in this study were from a laboratory colony originated in the 1950s from mosquitoes collected in Merced, CA (McAbee et al. 2004). Mosquitoes were kept in the laboratory at 27 ± 1 °C, under a photoperiod of 16:8 h light:dark and relative humidity of 75%.

Cloning and sequencing of CquiOR91

Twenty heads of last instar C. quinquefasciatus larvae were dissected and total RNA was extracted with Trizol (Thermo Scientific, Waltham, MA), following the manufacturer’s instructions. Reverse transcription and amplification of CquiOR91 were performed in one step using the OneStep RT-PCR kit (Qiagen, Valencia, CA). Gene-specific primers (0.6 µM final concentration) and larval RNA (1 µg) were used in a 50 µl reaction, following the manufacturer’s instructions. Reverse transcription and amplification of CquiOR91 were performed using the following conditions: 50 °C for 30 min (reverse transcription); 95 °C for 15 min (PCR activation); 40 PCR cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min 30 s followed by a final elongation step of 72 °C for 10 min. A PCR product at the expected size was purified from an agarose gel using the Qiaquick Gel Extraction Kit (Qiagen, Valencia, CA) and ligated into an EcoRV-digested pBluescript plasmid using T4 DNA ligase (Promega, Madison, WI). The ligation product was used to transform OneShot OmniMAX competent cells (Thermo Scientific, Waltham, MA). Twelve independent CquiOR91 clones were purified using the Plasmid Miniprep Kit (Qiagen, Valencia, CA) and sequenced (Davis Sequencing Inc, Davis, CA). The clones for both CquiOR91 versions, further named CquiOR91.1 and CquiOR91.2, were amplified from appropriate pBluescript plasmid templates using PfuUltra II Fusion DNA polymerase (Agilent Technologies, Santa Clara, CA) and transferred into a BamHI/EcoRI-digested pGEMHE plasmid (Liman et al. 1992; Hughes et al. 2010). The integrity of pGEMHE-CquiOR91.1 and pGEMHE-CquiOR91.2 sequences was verified by sequencing (Davis Sequencing Inc, Davis, CA). The sequences of CquiOR91.1 and CquiOR91.2 were released into the GenBank database under the accession numbers KX527860 and KX527861, respectively.

Primers used for RT-PCR:

CquiOR91f: 5′-ATGGATAAACTTCCGACGCGCAGTCC-3′

CquiOR91r: 5′-TTACTGTTGTAATTTCTTGAGCAGTGTGTA-3′

Primers used for subcloning into pGEMHE:

BamHI-CquiOR91f: 5′-TTAATGGATCCATGGATAAACTTCCGA CGCGCAG-3′

EcoRI-CquiOR91r: 5′-ATAATGAATTCTTACTGTTGTAATTTC TTGAGCA-3′

Cqui\Orco was isolated and inserted into the pGEMHE vector as described in our previous work (Hughes et al. 2010; Pelletier et al. 2010). Mutations were introduced using QuikChange Lightning kits (Stratagene). Each mutant construct was verified by sequencing.

Expression of ORs in Xenopus oocytes

Oocytes were surgically removed from mature X. laevis frogs and follicle cells removed by treatment with collagenase B (Roche Applied Science) for 2 h at room temperature. Capped cRNA encoding each OR subunit was generated using mMessage mMachine kits (Ambion). Oocytes were injected with 25 ng of cRNA encoding each subunit and incubated at 18 °C in Barth’s saline (in mM: 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 CaNO3, 0.41 CaCl2, 0.82 MgSO4, 15 HEPES, pH 7.6, and 150 µg/ml ceftazidime) for 2–6 days prior to electrophysiological recording.

Electrophysiology and data analysis

Odorant-induced currents were recorded under 2-electrode voltage clamp using an automated electrophysiology system (OpusXpress 6000A, Molecular Devices). Oocytes were perfused with ND96 (in mM: 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 5 HEPES, pH 7.5). Odorant stock solutions (usually 1 M) were prepared in dimethyl sulfoxide (DMSO). Odorants were diluted from stock into ND96 on the day of experimentation. Odorants were applied to the oocytes for 20 s at a flow rate of 1.65 ml/min, with extensive washing in ND96 (4.6 ml/min) between applications. Micropipettes were filled with 3 M KCl and had resistances of 0.2–2.0 M. The holding potential was −70 mV. Current responses, filtered (4-pole, Bessel, low pass) at 20 Hz (−3 db) and sampled at 100 Hz, were captured and stored using the OpusXpress 1.1 software (Molecular Devices).

Initial analysis was done using Clampfit 9 software (Molecular Devices). Concentration–response analysis was done by using PRISM 5 (GraphPad, San Diego, CA). Concentration–response curves were fit according to the equation: I = Imax/(1 + (EC50/X)n), where I represents the current response at a given concentration of odorant, X; Imax is the maximal response; EC50 is the concentration of odorant yielding a half-maximal response; and n is the apparent Hill coefficient.

Results

CquiOR91.2, but not CquiOR91.1, forms functional receptors

In order to test whether CquiOr91 is functionally similar to AgamOr40, we cloned the full-length receptor from a C. quinquefasciatus larval head RNA sample. Surprisingly, the sequencing of several independent CquiOR91 clones revealed 2 alleles of this OR differing at 9 residues (Figure 1), suggesting that these 2 forms might interact differently with odorant molecules. To understand the functional significance of these amino acid changes, we designed experiments to characterize the response profiles of both CquiOR91 alleles, hereafter named CquiOR91.1 and CquiOR91.2.

Figure 1.

Figure 1.

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Location of variable residues within a predicted secondary structure of CquiOR91.2. Snake plot representation of a predicted secondary structure of CquiOR91.2. The 9 positions that differ in CquiOR91.1 are indicated as the Or91.2 residue and Or91.1 residue inside a black circle. The image was constructed by hand, based on secondary structure analysis using Network Protein Sequence Analysis (Combet et al. 2000) and transmembrane domain estimation using TMpred (Hofmann and Stoffel 1993) and MEMSAT3 (Nugent and Jones 2009).

To functionally characterize the 2 forms of CquiOR91, we turned to the Xenopus oocyte expression system. Xenopus oocytes allow rapid, functionally accurate expression of a wide variety of receptors and channels, including insect ORs (Luetje et al. 2013). Odorant specificity information obtained for ORs expressed in Xenopus oocytes (Wanner et al. 2007; Nichols and Luetje 2010; Wang et al. 2010; Nichols et al. 2011) is comparable with odorant specificity information obtained for ORs expressed in the somewhat more “in vivo” Drosophila “empty neuron” expression system (Hallem et al. 2004; Hallem and Carlson 2006; Carey et al. 2010). Oocytes were injected with mRNA encoding each form into Xenopus oocytes, in combination with mRNA encoding the Culex OR coreceptor subunit (Cqui\Orco). Function was then assessed using 2-electrode voltage clamp electrophysiology. Based on the published characterization of Agam\Or40 (Xia et al. 2008), we used (+)-fenchone and (−)-fenchone as test ligands. Oocytes expressing CquiOR91.2 and Cqui\Orco responded robustly to application of 30 µM of each fenchone (Figure 2A). In contrast, oocytes injected with mRNA encoding CquiOR91.1 and Cqui\Orco failed to respond to 1 mM (−)-fenchone (n = 21) or to 1 mM (+)-fenchone (n = 6). The CquiOR91.2 + Cqui\Orco receptor displayed comparable sensitivities to (+)-fenchone and (−)-fenchone, with EC50’s of 107 ± 34 µM and 125 ± 43 µM, respectively (Figure 2F–G, Table 1). Similar to what has been reported for Agam\Or40 (Xia et al. 2008), we found that receptors formed by CquiOR91.2 could also be activated by DEET (Figure 2B,G, Table 1), albeit only at concentrations of 1 mM and greater. The high concentrations of DEET required to elicit a response from the CquiOR91.2 + Cqui\Orco receptor raised 2 concerns. First, we prepared DEET as a high concentration stock (1 M) in DMSO. Thus, a 10 mM DEET application in our experiment was also an application of 1% DMSO. However, we found that oocytes expressing the CquiOR91.2 + Cqui\Orco receptor, confirmed by robust response to 30 µM (−)-fenchone, did not respond to an application of 1% DMSO (Figure 2C, n = 8). A second concern is that the DEET responses we observed might be non-specific and not receptor mediated. For this reason, we also tested the highest concentration of DEET (10 mM) against sham (water) injected oocytes. Although oocytes expressing the CquiOR91.2 + Cqui\Orco receptor responded to 10 mM DEET with current amplitudes of 61 ± 13 nA (n = 4), application of 10 mM DEET to sham injected oocytes (Figure 2C) yielded current amplitudes of only 2.0 ± 0.5 nA (n = 7). The highly significant difference between these current responses (P = 0.0002, t-test) indicates that the DEET responses we observe with CquiOR91.2 + Cqui\Orco expressing oocytes are indeed receptor mediated. Thus, DEET is a weak agonist of the CquiOR91.2 + Cqui\Orco receptor. Oocytes injected with mRNA encoding CquiOR91.1 and Cqui\Orco failed to respond to 10 mM DEET (n = 6) or 30 mM DEET (n = 6) (Figure 6E).

Figure 2.

Figure 2.

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CquiOR91.2 forms functional receptors responsive to fenchone and DEET. (A) An oocyte expressing CquiOR91.2 + Cqui\Orco (left trace) responds to application of 30 µM (+)-fenchone and 30 µM (−)-fenchone. An oocyte injected with mRNA encoding CquiOR91.1 and Cqui\Orco (right trace) fails to respond to application of 1 mM (1000 µM) (+)-fenchone and 1 mM (−)-fenchone. (B) An oocyte expressing CquiOR91.2 + Cqui\Orco responds to 30 µM applications of (+)-fenchone and (−)-fenchone, as well as 1 mM (1000 µM) and 10 mM (10000 µM) applications of DEET. (C) An oocyte expressing CquiOR91.2 + Cqui\Orco responds to a 30 µM application of (−)-fenchone, but not to an application of 1% DMSO. (D) A water (sham) injected oocyte fails to respond to a 10 mM (10000 µM) application of DEET. (E) An oocyte injected with mRNA encoding CquiOR91.1 and Cqui\Orco fails to respond to 10 mM (10000 µM) DEET, 30 mM (30000 µM) DEET, or 30 µM (−)-fenchone. (F) Concentration–response analysis for activation of the CquiOR91.2 + Cqui\Orco receptor by (−)-fenchone. Current responses to each concentration of odorant (n = 8) were normalized to the response of the same oocyte to 30 µM (−)-fenchone. Curve fitting parameters are provided in Table 1. (G) Concentration–response analysis for activation of the CquiOR91.2 + Cqui\Orco receptor by (+)-fenchone and DEET. Current responses to each concentration of odorant (n = 4–7) were normalized to the response of the same oocyte to 30 µM (+)-fenchone. Curve fitting parameters are provided in Table 1.

Table 1.

Concentration–response parameters for activation Culex ORs

Receptor Ligand EC50 (µM) n H
CquiOR91.2 + Cqui\Orco (−)-fenchone 125 ± 43 0.73 ± 0.09
CquiOR91.2 + Cqui\Orco (+)-fenchone 107 ± 34 0.81 ± 0.11
CquiOR91.2 + Cqui\Orco Safranal 6 ± 2 0.8 ± 0.2
CquiOR91.1,C183Y + Cqui\Orco (−)-fenchone 148 ± 40 1.3 ± 0.3
CquiOR91.1,C183Y + Cqui\Orco Safranal 30 ± 7 1.1 ± 0.2
Cqui\Orco OLC12 82 ± 6 3.3 ± 0.7
CquiOR91.1 + Cqui\Orco OLC12 88 ± 5 4.0 ± 0.8
CquiOR91.2 + Cqui\Orco OLC12 35 ± 9* 1.8 ± 0.6
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nH, apparent Hill coefficient.

*Significantly different from Cqui\Orco value (P < 0.05, F-test).

A possible explanation for our failure to observe function for the CquiOR91.1 + Cqui\Orco receptor is that one or more of the 9 amino acid differences between CquiOR91.1 and CquiOR91.2 may have altered the ligand specificity of the subunit. That is, the CquiOR91.1 + Cqui\Orco receptor might have a ligand specificity that is distinct from that of the CquiOR91.2 + Cqui\Orco receptor. In an attempt to identify a ligand that can activate the CquiOR91.1 + Cqui\Orco receptor, we screened with a diverse panel of 155 odorants that broadly samples odor space (Li et al. 2012). The odorant panel was applied as a series of 8 mixtures (Table 2), with each mixture containing 17–20 compounds each at a concentration of 30 µM. Oocytes injected with mRNA encoding CquiOR91.1 and Cqui\Orco failed to respond to any of the 8 odorant mixtures, whereas oocytes expressing the CquiOR91.2 + Cqui\Orco receptor responded to several of the mixtures (Figure 3A). This result suggests that the CquiOR91.1 + Cqui\Orco receptor is either nonfunctional or is failing to form. However, because it is not feasible to screen every potential odorant ligand (of which there are millions), it is possible that a functional CquiOR91.1 + Cqui\Orco receptor is present and we have simply failed to apply the appropriate ligand (but see below).

Table 2.

Compounds contained within each screening mixture

Mixture 1 Mixture 2 Mixture 3
Compound CID Compound CID Compound CID
(−)-Ambroxide 10857465 (+)-Limonene 440917 (−)-Menthone 26447
(−)-Carvyl acetate 7335 2-Acetylpyrazine 2-acetylthiazole 30914 (+)-Citronellal 75427
(−)-Limonene 439250 2-Heptanol 520108 2-Acetylpyridine 14286
1-hexanethiol 8106 2-Methylanisole 10976 2-Ethoxythiazole 61809
2-Acetylfuran 14505 2-Pentanone 33637 2-Methyl butanal 7284
2-Methylpentanoic acid 7341 2,3,5,6-Tetramethylpyrazine 7895 2-Nonanol 12367
2-Pentanol 22386 4-Pentenoic acid 14296 2-Nonanone 13187
2,3-Dimethylpyrazine 22201 Allyl butyrate 61138 2-Phenylpropionaldehyde 7146
Benzyl salicylate 8363 Anisyl alcohol 16324 2,5-Dimethylpyrazine 31252
Butylamine 8007 Cinnamyl formate 7738 3-Heptanone 7802
Ethyl octanoate 7799 Isopentylamine 5354883 4-Allylanisole 8815
Furfuryl isopropyl sulfide 61282 Linalyl formate 7894 Allyl heptanoate 8878
Linalool 6549 Nerolidol 61040 Cinnamic acid 444539
Methyl phenylacetate 7559 Propyl disulfide 5284507 Cinnamyl alcohol 5315892
p-cresol 2879 Propyl formate 12377 Citral 638011
Phenethylamine 1001 Pyrrolidine 8073 Furfuryl heptanoate 557223
Piperidine 8082 α-Pinene 31268 Geranyl acetate 1549026
6654 Guaiacol 460
Propyl mercaptan 7848
trans-cinnamaldehyde 637511
Mixture 4 Mixture 5 Mixture 6
Compound CID Compound CID Compound CID
Submix 4.1 (−)-carvone 439570 Submix 6.1
 3-Heptanol 11520 (−)-isopulegol 170833  Citronellol 8842
 (+)-Isopulegol 1268090 (+)-dihydrocarvone 24473  Pinacol 6425
 3-Phenyl-1-propanol 31234 1-Nonanol 8914  (−)-carveol 7438
 trans, trans-2,4-octadienal 5283329 1,9-Nonanedithiol 248488  Eucalyptol 2758
 Cuminaldehyde 326 Thiolactic acid 62326  (−)-citronellal 443157
Submix 4.2 2-Pentylfuran 19602 Submix 6.2
 trans,trans-2,4-heptandienal 5283321 3-Octen-2-one 5363229  cis-4-decenal 5362620
 Phenylacetic acid 999 5-Methyl-2-phenyl-2-hexenal 5370602  Hydrocinnamaldehyde 7707
 Allyl tiglate 5364729 Benzenepentanol  Phenylacetaldehyde 998
 γ-Octalactone 7704 Amyl acetate 61523  trans-2-heptenal 5283316
 Heptyl butyrate 62592 Diphenyl ether 12348  Anisyl acetate 7695
Submix 4.3 Farnesal 7583 Submix 6.3
 Isobutyl phenylacetate 60998 Isobornyl propionate 5280598  Citronellyl valerate 61416
 Anisole 7519 Myrcene 89306  Heptyl acetate 8159
 2-Ethylpyrazine 26331 Nonyl acetate 31253  Prenyl acetate 14489
 (−)-Fenchone 82229 Octyl isobutyrate 8918  Theaspirane 61953
 (+)-carvone 16724 trans-2-hexenal 61024  Allyl-2-furoate 61337
Submix 4.4 trans-2-hexenoic acid 5281168 Submix 6.4
 (−)-β-pinene 440967 α-Ionone 5282707  (+)-Menthone 443159
 2-Isobutylthiazole 62725 5282108  2,4-Dimethylthiazole 10934
 2-Thiophenethiol 522674  3-Methylthio butanal 61845
 2,3-Pentanedione 11747  3-Penten-2-one 637920
 3-Nonanone 61235  Phenylacetaldehyde 998
 Skatole 6736
Mixture 7 Mixture 8
Compound CID Compound CID
Submix 7.1 Submix 8.1
 Farnesol 445070  Geraniol 637566
 4-Ethylguaiacol 62465  Carvacrol 10364
 (−)-Dihydrocarveol 443163  (S)-(−)-perillyl alcohol 369312
 (−)-Menthol 16666  Thymol 6989
 5-Methylfurfural 12097  2-Methyl pentanal 31245
Mixture 7 Mixture 8
Compound CID Compound CID
Submix 7.2 Submix 8.2
 Hydroxycitronellal 7888  o-methoxycinnamaldehyde 15173
 Safranal 61041  trans-3-hexenoic acid 5282708
 trans-2-methyl-2-pentenoic acid 5365909  Benzyl cinnamate 5273469
 Benzyl butyrate 7650  Ethyl butyrate 7762
 Diethyl succinate 31249  Hexyl isobutyrate 16872
Submix 7.3 Submix 8.3
 Isoamyl acetate 31276  p-tolyl acetate 8797
 Methyl-2-nonenoate 5368076  Isopropyl tiglate 5367745
 p-tolyl phenylacetate 60997  Terpinyl formate 16537
 Veratrole 7043  1,4-Cineole 10106
 d-camphor 159055  Dimethyl anthranilate 6826
Submix 7.4 Submix 8.4
 1-Octene-3-one 61346  2-Heptanone 8051
 4-Oxoisophorone 62374  6-Methyl-5-hepten-2-one 9862
 Allyl mercaptan 13367  Allyl sulfide 11617
 Ethyl 2-furoate 11980  Carvacrol 10364
 p-cymene 7463  Indole 798
 γ-Terpinene 7461
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Figure 3.

Figure 3.

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Screening with a broad panel of odorants identifies new ligands for CquiOR91.2, but fails to demonstrate function for CquiOR91.1. (A) Oocytes injected with mRNA encoding CquiOR91.1 and Cqui\Orco (top trace) or CquiOR91.2 and Cqui\Orco (bottom trace) were screened with 8 mixtures containing a total of 155 odorants. Each mixture contained 17–20 odorants, each at a concentration of 30 µM. The composition of each mixture is listed in the Table 2. (B) Oocytes expressing the CquiOR91.2 + Cqui\Orco receptor were screened with 4 submixtures derived from mixtures 6 and 7 (top trace) or mixtures 8 and 4 (bottom trace). Each odorant was present at a concentration of 30 µM. The composition of the submixtures is noted in Table 2. (C) An oocyte expressing the CquiOR91.2 + Cqui\Orco receptor was screened with the individual components (each at 30 µM) of submixtures 4.3 and 4.4: 3-nonanone (NON), 2,3-pentanedione (PEN), 2-isobutylthiazole (2-IBT), 2-thiophenethiol (TPT), (−)--pinene (PIN), isobutyl phenylacetate (IPA), anisole (ANI), 2-ethylpyrazine (2-EP), (+)-carvone (+CAR), (−)-fenchone (−FEN). (D) An oocyte expressing the CquiOR91.2 + Cqui\Orco receptor was screened with the individual components (each at 30 µM) of submixtures 6.1, 7.2, and 7.3: citronellol (COL), pinacol (PCL), (−)carveol (−CVL), eucalyptol (EUC), (−)-citronellal (CAL), hydroxycitronellal (HAL), safranal (SAF), trans-2-methyl-2-pentenoic acid (TMP), benzyl butyrate (BB), diethyl succinate (DES), isoamyl acetate (IA), methyl-2-nonenoate (MNN), p-toyl phenylacetate (PTP), veratrole (VER), d-Camphor (CAM). (E) Responses of oocytes expressing the CquiOR91.2 + Cqui\Orco receptor to 5 novel ligands (each at 100 µM) are presented as a percentage of the response of the same oocyte to 100 µM (−)-fenchone (mean ± SEM, n = 4). (F) An oocyte injected with mRNA encoding CquiOR91.1 and Cqui\Orco was screened with 1 mM applications of (−)-fenchone (-FEN), 2-isobutylthiazole (2-IBT), eucalyptol (EUC), veratrole (VER), d-Camphor (CAM), and safranal (SAF). (G) An oocyte injected with mRNA encoding CquiOR91.1 and Cqui\Orco was screened with 1 mM applications of (−)-carvone (−CAR), (+)-carvone (+CAR), 4-ethylphenol (4-EPH), p-cresol (CRE), 2-ethylphenol (2-EPH), and 4-methylcyclohexanol (MHC).

The responses of the CquiOR91.2 + Cqui\Orco receptor to multiple mixtures in Figure 3A suggested that novel odorant ligands were present in those mixtures. To identify individual compounds within the mixtures that are activating the CquiOR91.2 + Cqui\Orco receptor, we first subdivided the active mixtures (Figure 3B) and then screened individual components of the active submixtures (Figure 3C, D). We identified 5 novel odorant ligands for the CquiOR91.2 + Cqui\Orco receptor: 2-isobutylthiazole, veratrole, eucalyptol, d-camphor, and safranal (Figure 3C–E). Safranal yielded the largest responses, and concentration–response analysis showed that this compound was considerably more potent than either (+)-fenchone or (−)-fenchone at this receptor (EC50 = 6 ± 2 µM). Oocytes injected with mRNA encoding CquiOR91.1 and Cqui\Orco did not respond to a high concentration (1 mM) of these 5 novel odorant ligands (Figure 3F, n = 8). In addition, oocytes injected with mRNA encoding CquiOR91.1 and Cqui\Orco did not respond to a high concentration (1 mM) of 6 odorants previously shown to activate receptors formed by Agam\Or40 + Agam\Orco (Xia et al. 2008) (Figure 3G, n = 7).

A single amino acid change is responsible for the loss of function of CquiOR91.1 receptor

To explore possible reasons for our failure to observe function for the CquiOR91.1 + Cqui\Orco receptor, we generated a series of mutant CquiOR91.2 subunits, in which the residue at each of the 9 positions that differ between subunits was changed from what occurs in CquiOR91.2 to what occurs in CquiOR91.1. Each of these CquiOR91.2 mutants was coexpressed with Cqui\Orco in Xenopus oocytes and assayed for responsiveness to 1 mM (−)-fenchone. Eight of the 9 mutant versions of CquiOR91.2 were consistently able to form functional receptors and respond to (−)-fenchone (Figure 4A,B), suggesting that these residue changes were not critically involved in the apparent loss of function seen with CquiOR91.1. It is tempting to focus on the several mutations, such as M145I and N427Y, that appeared to dramatically decrease or increase the function or expression of the receptor. However, expression levels of exogenous receptors can vary dramatically among oocytes from a single batch, as well as among oocytes from different batches, whether from different frogs or from different surgeries from the same frog (Luetje et al. 2013). Thus, direct comparison of current amplitudes between oocytes should be avoided, if at all possible. Accordingly, we conclude simply that each of these 8 mutants was consistently functional. In contrast, oocytes injected with mRNA encoding CquiOR91.2,Y183C and Cqui\Orco consistently failed to respond to (−)-fenchone. To determine whether this single amino acid change was solely responsible for the lack of observable function for CquiOR91.1, we constructed a mutant version of CquiOR91.1 in which the cysteine at this position was changed to a tyrosine, as in CquiOR91.2. In contrast to wt CquiOR91.1, the mutant CquiOR91.1,C183Y was able to form a functional receptor when coexpressed with Cqui\Orco (Figure 4A,B). The CquiOR91.1,C183Y + Cqui\Orco receptor displayed sensitivities to (−)-fenchone and safranal (EC50’s = 148 ± 40 µM and 30 ± 7 µM, respectively) that were similar to what we observed for the CquiOR91.2 + Cqui\Orco receptor. These results suggest that the sole “defect” in CquiOR91.1 is the cysteine at position 183.

Figure 4.

Figure 4.

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Mutation analysis of CquiOR91.2 and CquiOR91.1 identifies position 183 as critical to CquiOR91 function. (A) Current recordings from oocytes injected with cRNA encoding the indicated wt or mutant receptor subunits (as well as Cqui\Orco) and challenged with 1 mM (−)-fenchone. (B) Current amplitudes of oocytes injected with cRNA encoding the indicated wt or mutant receptor subunits (as well as Cqui\Orco) and challenged with 1 mM (−)-fenchone are presented as a percentage of the response of wt CquiOR91.2 + Cqui\Orco to 1 mM (−)-fenchone (mean ± SEM). Mutants of CquiOR91.2: S38G (n = 14), I126T (n = 14), M145I (n = 8), Y183C (n = 31), S272P (n = 14), K276R (n = 14), N359D (n = 7), N414I (n = 8), N427Y (n = 8). CquiOR91.1 (n = 21), CquiOR91.1,C183Y (n = 8). The lack of function observed for CquiOR91.2,Y183C and wt CquiOR91.1 is represented by “0.”

In a predicted secondary structure of the CquiOR91 subunit (Figure 1), residue 183 is located in the second extracellular loop, a region thought to play a role in the structure of the ligand-binding site of these receptors (Xu and Leal 2013). Thus, it may be that the CquiOR91.1 subunit is forming a receptor with Cqui\Orco but that this receptor has a defective ligand-binding site. To test this possibility, we took advantage of our previous findings, with ORs from several species, that heteromeric OR complexes are more sensitive to activation by Orco agonists than are the Orco homomers (Chen and Luetje 2012, 2013, 2014). When we used the Orco agonist OLC12 to activate the homomeric Cqui\Orco receptor, we obtained an EC50 of 82 ± 6 µM (Table 1). The CquiOR91.2 + Cqui\Orco receptor was significantly more sensitive to OLC12, displaying an EC50 of 35 ± 9 µM (P = 0.0109, F-test). In contrast, oocytes injected with mRNA encoding the CquiOR91.1 subunit and Cqui\Orco responded to OLC12 with an EC50 (88 ± 5 µM) that was indistinguishable from the Cqui\Orco homomer (P = 0.5507, F-test). This result suggests that CquiOR91.1 is not present in a complex with Cqui\Orco and that the failure to yield odorant responses is not merely due to a defect in the binding site.

Discussion

The identification of a single amino acid change responsible for the loss of function of CquiOR91 is intriguing. The CquiOR91 reference sequence in VectorBase (CPIJ009579) harbors the tyrosine residue in position 183, similar to the functional CquiOR91.2 allele, suggesting that the functional form might be predominant in nature, at least in the Johannesburg strain that was used to establish the genome sequence. The possibility that the nonfunctional allele is derived from mutation events that occurred exclusively in the laboratory colony is highly unlikely because CquiOR91.1 and CquiOR91.2 display 8 polymorphic residues, in addition to the variant at position 183. On the other hand, the presence of the nonfunctional allele in our mosquito colony might be a “genetic drift” effect due to the small size of the colony, possibly facilitated by the artificial rearing conditions.

The functional characterization of CquiOR91.2 provides another instance of orthologous mosquito ORs that retain similar functions across species. With some overlap between our screening panel and the panel used to characterize Agam\Or40 (Xia et al. 2008), we can compare response profiles of the 2 ORs. Both ORs responded to fenchone and DEET (although the CquiOR91.2 response to DEET was quite weak), but failed to respond to indole, 2-acetylpyridine, 2-acetylthiazole, 2-ethoxythiazole, isoamyl acetate, 6-methyl-5-hepten-2-one, and 2-nonanone. There were also some differences between the 2 receptors, with Agam\Or40 receptors responding modestly to p-cresol and carvone, whereas CquiOR91.2 receptors did not respond to these odorants. Also, CquiOR91.2 receptors responded modestly to 2-isobutylthiazole, whereas Agam\Or40 receptors did not. The similarity between the response profiles of CquiOR91.2 and Agam\Or40 suggest that different mosquito species are equipped with ORs that are able to detect a common set of ligands that are important for survival at the larvae stage. Our screening experiments also identified a novel ligand, safranal, as the most potent agonist for CquiOR91.2. At present, it is not known whether safranal is also a potent ligand for Agam\Or40 or Aaeg\Or40.

CquiOR91.2 also displayed functional similarity to Agam\Or40 in responding to the insect repellent DEET, albeit only at very high concentrations. The complete mode of action of DEET is still a matter of considerable debate (Ditzen et al. 2008; DeGennaro et al. 2013; Xu et al. 2014; Silbering et al. 2016). DEET is both a spatial and a contact repellent, acting as both an odorant and a tastant, most likely affecting multiple receptors. Recently, a DEET-sensitive OR (Cqui\Or136) was identified in the genome of the southern house mosquito (Xu et al. 2014). In contrast to CquiOR91, which is expressed in larvae and has orthologs in the Aedes and Anopheles mosquitoes, Cqui\Or136 is expressed in adult female antennae and has no apparent orthologs in the other 2 mosquito species. It is likely that additional DEET-sensitive ORs, in addition to DEET-sensitive GRs, remain to be discovered.

Funding

This work was supported by grants from the National Institutes of Health (RO1 DC011091 to C.W.L. and R01 AI095514 to W.S.L.). D.T.H. was supported, in part, by T32 NS007044.

Acknowledgments

We thank A. Castro and B. Sherman for oocyte preparation.

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