An odorant receptor from Anopheles sinensis in China is sensitive to oviposition attractants

Anopheles sinensis (laboratory-susceptible strain) larvae and pupae were reared on yeast powder, and adults were maintained on a 10% sugar solution at 25–27 °C and 70–80% relative humidity with a photoperiod of 12:12 h. Three-day-old adult females were blood-fed on a human volunteer arm using standard protocols [17].

Predicted amino acid sequences of An. sinensis (ASIS023681-RA) [18], Anopheles funestus (KF819859), Anopheles gambiae (AGAP002560-RA) and Culex pipiens quinquefasciatus (DQ231246) Orco orthologs were obtained from VectorBase. Primers (Additional file 1: Table S1) used for two-step RT-PCR were first designed based on these sequences using primer 5.0 to amplify partial gene sequences of AsinOrco and AsinOR10. Total RNA extraction from female adult mosquitoes (3–7 days old) and cDNA synthesis were performed using an RNeasy Mini Kit (QIAGEN, Hilden, Germany), the TURBO DNA-free™ Kit (Ambion, Carlsbad, CA, USA) and TaKaRa PrimeScript™ RT-PCR Kit (Takara, Otsu, Shiga, Japan) following the manufacturers’ instructions. Gene-specific primers (Additional file 1: Table S1) were then designed for 5′- or 3′-end rapid amplification of cDNA ends (RACE) to amplify full-length coding sequences using a SMARTer™ RACE cDNA amplification kit (Clontech, Mountain View, CA, USA).

Nested RT-PCR primers (Additional file 1: Table S1) were designed based on the predicted amino acid sequences of An. sinensis (ASIC007209-RA) [18], Culex pipiens (FJ008065), Culex quinquefasciatus (GU945397), Anopheles quadriannulatus (FJ008069), An. gambiae (AGAP009520-RA) and Anopheles stephensi (FJ008074) OR10 orthologs. PCR was carried out using TaKaRa Tks Gflex DNA Polymerase (Takara, Otsu, Shiga, Japan). PCR amplification products were examined by 1.5% agarose gel electrophoresis and verified by DNA sequencing (Invitrogen, Shanghai, China). The obtained sequences were compared with predicted AsinORs and AgamORs using DNAMAN.

Amino acid sequences of ORs were aligned using the program ClustalW, and the neighbor-joining tree was built using the MEGA 5.0 program [19]. The membrane topology of the OR sequences was predicted using the HMMTOP (version 2.0) and TMHMM (version 2.0) [20] servers.

The full-length coding sequences (CDSs) of AsinORs were cloned into the pME18s mammalian expression plasmid [9] using specific primers (Additional file 1: Table S1). The DsRed coding sequence was amplified from pIRES2-DsRed plasmids (Clontech, Mountain View, CA, USA) using primers containing the appropriate restriction sites. AsinORs were cloned into the pME18s plasmid in-frame with the DsRed coding sequence [8]. HEK293 (human embryo kidney 293) cells (purchased from the Chinese Academy of Sciences) were cultured in an incubator at a constant temperature of 37 °C with 5% CO₂ and transiently transfected with AsinORs using Lipofectamine^® 2000 Reagent (Invitrogen, Carlsbad, CA) [21]. Expression of ORs was confirmed by RT-PCR after 24 h; subcellular location analysis and western blotting were performed after 48 h. The two-step RT-PCR primers and nested RT-PCR primers are provided in Additional file 1: Table S1. Cells were lysed with RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 0.1% Triton X-100, 1% Nonidet P-40). The lysates were mixed with in SDS–PAGE buffer (62.5 mM Tris, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue), heated at 95 °C for 5 min, separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Immobilon^TM-P, Millipore). The blot was washed with TBST, incubated with 5% skim milk for 60 min, and incubated overnight with an anti-RFP antibody (Abcam, Cambridge, US) raised in mice at a dilution ratio of 1:1000 in 1 × PBS or anti-GAPDH antibody (Abcam, Cambridge, US) at 1:3000 dilution at 4 °C. The blot was then incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1:4000) (Bethyl Laboratories, Montgomery, TX, USA) at room temperature for 90 min.

Forty-eight hours after transfection, AsinOR-expressing cells were rinsed three times with HBSS. Fluo4-AM (Dojindo Laboratories, Tokyo, Japan) at a concentration of 2 μM was added, and the cells were incubated for 30 min at 37 °C in the dark. The cells were rinsed three times with HBSS before the addition of fresh HBSS (containing Ca²⁺) [8] and tested using a panel of odorants, including indole, 1-octen-3-ol, 1-methylindole, 3-methylindole, 2-methylphenol, 2,3-butanedione, 2-ethylphenol, and dimethyl sulfoxide (Sigma). All odorants (≥ 98% pure) were dissolved in DMSO and added to a final concentration of 10⁻⁶ M.

Fluorescence images were acquired using a laser scanning confocal microscope (Olympus, Japan). The Ca²⁺ level is represented as relative fluorescence change (ΔF/F₀), where ΔF is the difference in peak fluorescence caused by stimulation and F₀ is the baseline fluorescence [22, 23]. Baseline fluorescence was measured 100 s prior to adding the chemicals. Responses were quantified by the mean values of the maximal elevations (ΔF/F₀) [8]. Each odorant was assayed in triplicate per dish, and at least seven cells per dish were selected randomly. All assays were performed in triplicate.

Full-length coding sequences for AsinOrco and AsinOR10 were successfully obtained based on bioinformatics and homologous genes. AsinOrco, which is 1437 bp in length and encodes 479 amino acids, exhibits 96.66% sequence identity with predicted AsinOrco (98.96%) and AgamOrco (90.61%). Similarly, AsinOR10, which is 1125 bp in length and encodes 375 (93.35%) amino acids, shares 100% and 80.59% identity with predicted AsinOR10 and AgamOR10, respectively. An alignment of AsinOrco (97.49%) and AsinOR10 (90.37%) amino acid sequences with related sequences in An. gambiae is shown in Figs. 1, 2. In general, ORs display a high level of divergence [24]. An interesting phenomenon is that the ORs from different species have very high sequence conservation.

To explore relationships among ORs from different species, phylogenetic tree analysis was carried out using similar OR sequences, mainly including AgamORs, AaegORs and CquiORs. The results revealed the existence of different subgroups (Fig. 3). For this study, AsinOrco and AgamOrco, AfunOrco, AalbOrco, AaegOrco, CquiOrco and CppOrco were found to be clustered together. This finding indicates that AsinOrco belongs to the coreceptor subfamily, whereas AsinOR10, which is identified as a conventional odorant receptor, clusters with the OR2-10 subgroup. Among them, AsinOrco and AsinOR10 display the highest identity with AgamOrco/AfunOrco and AgamOR10/AsteOR10, respectively. Significantly, with the exception of OR7 orthologs, OR2 and OR10 are the most conserved ORs in the phylogenetic tree. This sequence conservation suggests that OR10 may show an odorant-induced response profile similar to that of OR2. These interesting phenomena encouraged us to examine the odorant response profile of AsinOR10.

Membrane topology predictions for AsinOrco and AsinOR10 revealed that these receptors belong to the seven-transmembrane (TM) protein family with an intracellular amino-terminus (Fig. 4). Analysis of the primary amino acid sequence of AsinOrco shows that it contains a putative calmodulin (CaM)-binding site (³²⁸SAIKYWVER³³⁶) identified in DmelOrco (³³⁶SAIKYWVER³⁴⁴) and in AalbOrco (³²⁹SAIKYWVER³³⁷) [8]; in contrast, AsinOR10 does not have this putative CaM-binding site or channel gate sequences. The observed sequence conservation supports our hypothesis that AsinOrco may form a channel gate, as reported for DmelOrco [25, 26], and that it may form complexes involved in odour signal transduction [20, 25].

AsinOR transcripts were detected in HEK293 cells at 24 h (Fig. 5a), and corresponding proteins at approximately 78 kDa and 67 kDa (Fig. 5b) were identified by western blotting at 48 h. The previous study [8] found that individual OR proteins respond weakly to certain test chemicals but that HEK293 cells coexpressing Orco and OR respond strongly. Therefore, HEK293 cells coexpressing AsinOrco and AsinOR10 were screened using a panel of odorants at a final concentration of 10⁻⁶ M (Fig. 6) in calcium-imaging experiments. The strongest fluorescence was elicited by 3-methylindole (skatole) (measured as the relative fluorescence change, ΔF/F0). Interestingly, except for 3-methylindole (F_{(7, 461)} = 120.240, P < 0.005; Dunnett T3 vs DMSO, 3-methylindole: P < 0.005; indole: P = 1.000; 1-octen-3-ol: P = 1.000; 1-methylindole: P = 0.188; 2-methylphenol: P = 0.320; 2,3-butanedione: P = 1.000; 2-ethylphenol: P = 1.000), the receptors showed no significant differences in their responses to other odorants compared to dimethyl sulfoxide (DMSO). The study interprets these results to indicate that AsinOR10 has high sensitivity for 3-methylindole but very low sensitivity for indole and other methylindoles, including 1-methylindole.