Expressional and functional interactions of two Apis cerana cerana olfactory receptors

Odors

All odorants used in the present study were purchased from (Sigma-Aldrich, St. Louis, MO, USA) and were of the purest grade (>95% pure). Stock solutions (100 mM) of the odorants were prepared using dimethyl sulfoxide (DMSO) and stored at −20 °C. For each assay, odorant solutions were freshly diluted from the stock solution to the desired concentration in DMSO. Fluo-4-(acetoxymethyl) ester (Fluo-4 AM) (excitation at 494 nm, emission at 516 nm), obtained from (Beyotime, Shanghai, China) as a lyophilized powder, was diluted to 1 mM using DMSO and stored at −20 °C. The composition of the calcium assay buffer was as follows: 21 mM KCl, 12 mM NaCl, 18 mM MgCl₂, 3 mM CaCl₂, 170 mM D-glucose, 1 mM probenecid (Sigma-Aldrich, St. Louis, MO, USA), and 10 mM piperazine-1,4-bisethanesulfonic acid. The pH of the buffer was adjusted to 7.2, and the buffer was filter-sterilized (using a 0.22 µm filter) prior to use.

Vector construction

The pIB-AcerOr1/pIB-AcerOr2 plasmid constructs containing intact open reading frames, which were amplified with specific primers containing BamHI and EcoRI (NEB, Beverly, MA, USA) sites for the A. cerana cerana ORs AcerOr1 and AcerOr2 and cloned in the multiple cloning site of the pIB/V5-His vector (Invitrogen, Carlsbad, CA, USA), were used to generate the final transformation plasmids by restriction digestion with BamHI and EcoRI (NEB, Beverly, MA, USA). The specific primer sequences were as follows: AcerOr1 F: 5′-CGCGGATCCATGGAAAATACCACGAATTATCGTA-3′, AcerOr1 R: 5′-CCGGAATTCTACCGTCATTGCACGCAGAA-3′, AcerOr2 F: 5′-CGCGGATCCATGATGAAGTTCAAGCAACAGGG-3′, AcerOr2 R: 5′-CCGGAATTCCTTCAGTTGCACCAACACCA-3′.

Cell culture and transfection of Sf9 cells

Spodoptera frugiperda Sf9 cells were purchased from the Chinese Academy of Sciences Cell Bank (Beyotime, Shanghai, China) were maintained as an adherent culture in Sf-900 III serum-free medium (SFM; Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou, China) and 100 μg mL⁻¹ penicillin-streptomycin at a constant temperature of 28 °C in a humidified incubator (Thermo Scientific, Cornelius, OR, USA) in the absence of CO₂ in T-25 tissue culture flasks (Corning Inc., Corning, NY, USA). Sf9 cells were grown to approximately 80–90% confluence as observed under a light microscope. Cells were dislodged from the flask by washing with the media contained in the flask. In total, 1 × 10⁶ Sf9 cells were suspended in 2 mL of Sf-900 III SFM in each well of a Nunclone six-well tissue culture plate (Corning Inc., Corning, NY, USA). Confluent cells (80–90%) were transiently transfected with 2.0 μg pIB-AcerOr1/pIB-AcerOr2 using 8 μL of Cellfectin II reagent (Invitrogen, Carlsbad, CA, USA) in six-well plates according to the manufacturer’s instructions. The medium containing plasmid DNA and Cellfectin II was removed after incubation of the cells with a DNA/Cellfectin II mix for 3–5 h. The cells were washed twice with fresh Sf-900 III SFM and overlaid with 2 mL of fresh SFM. G418 was used to select stably transfected cell lines, and the optimum G418 concentration for screening the stable cell lines was 400 μg mL⁻¹. After incubation for 48 h, assays were performed.

Western blot and immunofluorescence analysis

To acquire polyclonal antigens (pAb_AcerOr1 and pAb_AcerOr2) of high titer and specificity against OR AcerOr1 and AcerOr2, the primary amino acid sequence and secondary protein structure information for AcerOr1 and AcerOr2 in NCBI GenBank was used, and one sequence-specific polypeptide was obtained using the bioinformatics software BLASTn, BLASTx, ExPASy, DNASTAR, and ANTHEPROT. These programs were used to analyze the amino acid sequence features of AcerOr1 and AcerOr2 proteins, including their hydrophilicity, flexibility, surface probability and antigenicity, and their secondary structures, and to find the antigenic peptides AcerOr1 ENTTNYRNIHYKSD (14 aa) and AcerOr2 NARYHQIAVK (10 aa). An AcerOrs antibody made by AbMax (AbMax Biotechnology Co., Ltd., Beijing, China) was used for western blot analysis and immunostaining to confirm the expression of AcerOr1 and AcerOr2.

Goat anti-Rabbit IgG, Alexa Fluor 488/594, and 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) were used to stain AcerOr1/AcerOr2 in transfected Sf9 cells grown on poly-l-lysine-coated coverslips placed in six-well plates. Thereafter, the medium was removed from the wells, and the cells were washed with phosphate-buffered saline and fixed with 4% (v/v) paraformaldehyde for 30 min at 25 °C. They were then treated with 5% bovine serum albumin for 1 h at 25 °C. Subsequently, the sections were incubated with rabbit anti-AcerOr (1:2,000 dilution) polyclonal antibody overnight at 4 °C. The secondary antibody (goat anti-Rabbit Alexa Fluor 488 or Alexa Fluor 594; 1:10,000) was applied for 2 h at 37 °C. Next, cells were incubated with 1 mL of DAPI (1:10,000). The coverslips with the stained cells were removed for analysis using immunofluorescence microscopy. Images were analyzed using the ImageJ software (National Institute of Health, Bethesda, MD, USA).

For western blotting, protein was extracted from cells expressing plasmids and transfected with either AcerOr1 or AcerOr2 or co-transfected with AcerOr1 and AcerOr2 using a cell lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). The extracted proteins (100 μg per sample) were separated by 12% SDS-PAGE and transferred onto a nitrocellulose filter membrane (Boster, Wuhan, China). Membranes were blocked for 1.5 h at 25 °C in 5% skim milk (Boster, Wuhan, China), washed with Tris-buffered saline containing Tween-20 (TBST, pH 8.0), and incubated overnight at 4 °C with rabbit polyclonal anti-AcerOr, mouse anti-His-tagged (1:1,000 (v/v)) (BioWorld, Minneapolis, MN, USA) and mouse anti-β-actin (1:500 (v/v)) (Boster, Wuhan, China) antibodies. Thereafter, membranes were washed with TBST and incubated with the secondary antibodies, namely horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:5,000 (v/v)) (Boster, Wuhan, China) and goat anti-mouse (1:2,000 (v/v)) IgG (Boster, Wuhan, China), respectively, for 2 h at 25 °C. Finally, membranes were washed three times with TBST. Bands were detected using Super ECL Plus detection reagent (Boster, Wuhan, China), and the western blot signal was analyzed using Image Lab (Bio-Rad Laboratories, Hercules, CA, USA) and Image J 1.49.

Ca²⁺ imaging

To identify candidate ligands for selected ORs, we tested 32 compounds (most of which were volatile compounds from host plants, including aldehydes, alcohols, monoterpenes, benzoates, and sesquiterpenes) at a final concentration of 10⁻⁶ M by Ca²⁺ imaging in an in vitro cell expression system. Thereafter, we determined the concentration-response curves for ten compounds (selected from the 32 compounds) and calculated their half-maximal effective concentration (EC₅₀) values. After the cells were transfected for 48 h, the medium was removed and the cells were washed three times with Hank’s balanced salt solution (without Ca²⁺). The cells were subsequently cultured at 37 °C in the dark for 30 min in the presence of 2 μmol L⁻¹ Fluo-4-AM (Beyotime, Shanghai, China) and were stimulated by the chemical odorants. Each test chemical ligand was applied to Fluo-4-loaded Sf9 cells expressing AcerOr1 at a final concentration of 10⁻⁶ M, and the increase in fluorescence caused by the substrate was measured and expressed as a fraction of the fluorescence elicited by the calcium ionophore ionomycin. The Ca²⁺-free solution used was Dulbecco’s phosphate buffered saline supplemented with 0.4 mM ethylene glycol tetra-acetic acid (EGTA). Fluorescence was measured using excitation and emission wavelengths of 494 and 516 nm, respectively, and the results were recorded by a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). The formula used for calculating the free intracellular Ca²⁺ concentration was as follows: $$\left[{\text{Ca}}^2{}^{+}\right]i=K_d\left(\frac{F-F_{\text{min}}}{F_{\text{max}}-F}\right)$$ where F_min and F_max are the minimum fluorescence values under Ca²⁺-saturating conditions in the presence of 5 μM A23187 (a Ca²⁺-ionophore) and the maximum fluorescence values under zero-Ca²⁺ conditions when 4 mM EGTA was used in combination with 5 μM A23187, respectively. K_d is the dissociation constant of Fluo-4/Ca²⁺ (360 nM).

EC₅₀ values were determined using GraphPad Prism 6.0 (GraphPad; San Diego, CA, USA). The formula used for calculating the concentration-response using a 4-parameter logistic model was as follows: $$Y=\text{Bottom}+\frac{(\text{Top}-\text{Bottom})}{1+{10}^{{\left({\text{LogEC}}_{50}-X\right)}^{*}\text{HillSlope}}}$$ where Y and X are the response showing a sigmoid shape and the logarithm of the concentration. EC₅₀ is the concentration of odorant yielding 50% of its maximal effects, and HillSlope is the slope parameter. Residual standard deviation, the coefficient of determination (Xu et al., 2012), and 95% confidence intervals were calculated to verify that the fitted curve was correct.

Electroantennography

The level of OR expression in antenna olfactory neurons can be indirectly measured by recording the EAG responses of the isolated antennae to the corresponding odorant ligand. We compared data previously obtained by Ca²⁺ imaging in vitro to determine whether these odorants can cause electrophysiological responses in the antennae with physiological activity. Based on the results of the Ca²⁺ assay, three volatile compounds (VUAA1, eugenol, and linolenic acid) were used to record antennal responses. The compounds were dissolved and diluted in liquid paraffin to final concentrations of 0.1, 1, 10, 100, and 500 μg μL⁻¹. Pure liquid paraffin wax was used as a blank, and results were calculated relative to the blank. The antennae were carefully cut at the base and placed into EAG electrode probes (Syntech, Hilversum, The Netherlands) with a drop of Spectra 360 electrode gel (Parker Lab, Inc., Fairfield, NJ, USA). Filter paper strips (5 × 50 mm) were loaded with 20 μL of the different test solutions and inserted into glass Pasteur pipettes and served as sources of stimuli. Humidified airflow was delivered at a constant rate of 700 mL min⁻¹ by an air stimulus controller CS-55 (Syntech, Kirchzarten, Germany). Odor stimuli were administered three times at 2 mL s⁻¹ for 0.5 s at 30 s intervals. EAG recordings of antennal responses to each stimulus were documented as voltage waveforms using an IDAC-4 computer-operated amplifier controller (Syntech, Kirchzarten, Germany), and the data were analyzed with the EAGPro software (Syntech, Kirchzarten,Germany). A newly prepared antenna was used for each recording. A dose-response curve was plotted using the EAG recordings (in mV) for each concentration.

Statistical analysis

The TMDs were predicted using TTHMM server v.2.0 and HMMTOP. Data were analyzed with SPSS v17.0 (SPSS Inc., Chicago, IL, USA) and expressed as the mean ± standard error (SEM). t-tests, ANOVAs, and Duncan’s multiple range tests were used to determine whether differences in the mRNA and protein levels or the EAG responses of antennae were significantly different among treatments. In all cases, statistical significance was tested at the 0.05 level.

Membrane topology analysis of the AcerOrs

We first analyzed the primary amino acid sequence of the A. cerana cerana AcerOrs protein. We identified that AcerOr1 did not have the putative CaM binding site and gate sequences, and we thus selected the sequence ENTTNYRNIHYKSD (14 aa) in the N-terminal hydrophobic area as the multiple antigen peptide of AcerOr1 (Fig. 1A). A candidate calmodulin (CaM)-binding amino acid motif ³²⁸SAIKYWVER³³⁶ within the second intracellular loop (ICL2) of the AcerOr2 protein and, the sequence NARYHQIAVK (10 aa) in the ICL1 domain served as the multiple antigen peptide of AcerOr2 (Fig. 1B).

Heterologous expression and localization of AcerOr1 and AcerOr2 in Sf9 cells

To confirm that ORs were successfully transfected in the Sf9 cells, the cells was stained with anti-AcerOr1 or anti-AcerOr2 followed by goat anti-rabbit Alexa Fluor 488 or 594. The results revealed that both AcerOr1 and AcerOr2 were expressed and localized to the plasma membrane of Sf9 cells (Fig. 2A), as pIB/V5-His-transfected Sf9 cells showed no immunostaining. Western blotting of Sf9 cell extracts using an anti-AcerOr1 and anti-AcerOr2 antibody revealed a specific band of approximately 50 and 56 kDa in Sf9 cells transfected with pIB/V5-AcerOr1 and pIB/V5-AcerOr2, but no specific bands in the Sf9 cells (negative control) or pIB/V5-His-transfected Sf9 cells (Fig. 2B). These results confirmed successful construction of the recombinant plasmids and expression of the corresponding OR in the Sf9 cells after in vitro transfection.

Screening of specific ligands of AcerOrs

To test the potential functional activity of AcerOr during the olfaction process, odorant ligand binding is essential. The transfected cells were tested against a panel of 32 odorants (Fig. 3). The control cells transfected with the pIB/V5 empty vector were indifferent to all odorants tested (Fig. 3A). Nine of the 32 compounds, including eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol, elicited responses from AcerOr1-expressing cells when administered at the high concentration of 10⁻⁶ M. Each of these odor compounds activated AcerOr1 to differing levels as reflected by differing fluorescence intensities (Fig. 3B). AcerOr2 expressed alone did not show responses to any floral odorants except its agonist VUAA1, which yielded the greatest increase in intracellular Ca²⁺, whereas those expressing AcerOr1 alone were sensitive to all nine volatile compounds mentioned above and VUAA1 (Fig. 3).

To examine the sensitivity of AcerOr for the above-described selected ligands, calcium assays were conducted for a range of ligand concentrations to support the construction of concentration-response curves with calculated half-maximal effective concentration (EC₅₀) values. Ligand concentrations were reduced by 10-fold starting at 10⁻⁵ M. We then determined the activity of the ten compounds in more detail by constructing concentration-response curves for activation of AcerOr1 and AcerOr2 alone (Fig. 4). For AcerOr1 activation, the EC₅₀ values ranged from 10⁻⁷ to 10⁻⁸ M for VUAA1, eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol; for AcerOr2 activation, the EC₅₀ value of VUAA1 was (6.621 ± 0.26) × 10⁻⁸ M (Fig. 4; Table 1).

Electrophysiological response of Apis cerana cerana antennae

The selected odorants were tested for their the ability to elicit EAG responses in the antennae of A. cerana cerana, and three floral volatiles (VUAA1, eugenol, and linolenic acid) caused irritation and elicited EAG responses (Fig. 5). All three compounds showed a dosage-dependent increase in EAG response, and the highest effect was observed at a high concentration of 500 μg μL⁻¹ of compound (18.71 ± 1.45, 9.92 ± 0.58, 10 ± 0.54 mV, respectively.) compared with 0.1, 1, 10, and 100 μg μL⁻¹, (P < 0.05; Fig. 5). These results were consistent with those for Ca²⁺ imaging.