A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid

Abstract

By using a functional genomics approach, we have identified a honey bee [Apis mellifera (Am)] odorant receptor (Or) for the queen substance 9-oxo-2-decenoic acid (9-ODA). Honey bees live in large eusocial colonies in which a single queen is responsible for reproduction, several thousand sterile female worker bees complete a myriad of tasks to maintain the colony, and several hundred male drones exist only to mate. The "queen substance" [also termed the queen retinue pheromone (QRP)] is an eight-component pheromone that maintains the queen's dominance in the colony. The main component, 9-ODA, acts as a releaser pheromone by attracting workers to the queen and as a primer pheromone by physiologically inhibiting worker ovary development; it also acts as a sex pheromone, attracting drones during mating flights. However, the extent to which social and sexual chemical messages are shared remains unresolved. By using a custom chemosensory-specific microarray and qPCR, we identified four candidate sex pheromone Ors (AmOr10, -11, -18, and -170) from the honey bee genome based on their biased expression in drone antennae. We assayed the pheromone responsiveness of these receptors by using Xenopus oocytes and electrophysiology. AmOr11 responded specifically to 9-ODA (EC50=280+/-31 nM) and not to any of the other seven QRP components, other social pheromones, or floral odors. We did not observe any responses of the other three Ors to any of the eight QRP pheromone components, suggesting 9-ODA is the only QRP component that also acts as a long-distance sex pheromone.

Fig. 1.

The drone olfactory system is sexually dimorphic. Morphology of drone (A–D) and worker (E–H) honey bee olfactory systems. (A) Frontal view of the drone head illustrating the enlarged eyes and antennae and reduced mandibles compared with the worker in E. SEM photograph of a single antennal segment from drone (B) and worker (F) antennae illustrating the higher density of poreplate sensilla and reduced number of trichoid sensilla on the surface of drone antennae. (C and G) Enlarged view of the drone and worker antennal surface (×1,950 magnification). Photographs of drone and worker antennal surfaces were taken with a Philips XL30 ESEM-FEG microscope (FEI, Hillsboro, OR; Imaging Technology Group, Beckman Institute at UIUC). (D) A reconstructed drone antennal lobe (frontal view) illustrating four enlarged macroglomeruli, MG1–4. MG2 responds to antennal stimulation with 9-ODA (36). 3D reconstruction of the drone antennal lobe was created with AMIRA for Windows 3.0 software (TGS, San Diego CA). (H) Frontal view (2D transparent projection) of a worker antennal lobe (tetramethylrhodamine dextran-stained) illustrating isomorphic glomeruli. Microscopy and image analysis were performed with a Leica TCS SP2 confocal microscope (Imaging Technology Group, Beckman Institute at UIUC) with green (543-nm) and red (633-nm) helium/neon laser lines. Images (1,024 × 1,024 pixels) were acquired by using a ×10 or ×20 objective.

Fig. 2.

Four candidate sex pheromone receptors are expressed at higher levels in drone antennae. Sex-biased expression of olfactory-related genes in drone and worker antennae determined by microarray analysis (two-way dye-swap design, n = four replicates). The volcano plot indicates that the majority of the genes were not differentially expressed (fold change in expression was >0.5 and <2.0, and the P values were >0.05). Ten genes were expressed at higher levels in worker antennae (upper left quadrant, P < 0.05, and fold change in expression <0.5), and six were expressed at higher levels in drone antennae (upper right quadrant, P < 0.05, and fold change in expression >2.0). GenBank accession nos.: AAR2 (XM394372), CEst1 (AY647436), CSP6 (NM1077819), Cyp6BE1 (XM624792), Lip1 (NM1011630), OBP2, -4, -11, -13, -14, and -19 (NM1011591, NM1011589, NM1040226, NM1040224, NM1040223, and NM1040209); Ors 10, 11, 18, 151, and 170 are published (25).

Fig. 3.

Three of the four candidate sex pheromone receptors group closely together in the honey bee phylogenetic tree. Drone-to-worker ratio of AmOr genes (n = 43) expressed in antennae, determined by qPCR. AmOr2, -4, -5, -6/-7, -8 to -25, -29, -30, -35, -42, -49, -50, -60, -63, -68, -78, -81, -89, -95, -109, -126, -132, -149 to -152, and -170 are depicted sequentially on the x axis. Cycle threshold (CT) values for each gene expressed in drone and worker antennae were normalized to the control gene AmRPS8 before calculating the drone-to-worker ratio (y axis). The phylogenetic relationships of AmOr4–30 are represented by a neighbor-joining tree as described in ref. .

Fig. 4.

9-ODA activates AmOr11 + AmOr2. (A) Oocytes injected with RNA encoding AmOr10 + AmOr2, AmOr11 + AmOr2, AmOr18 + AmOr2, or AmOr170 + AmOr2 are challenged with 100 μM HOB, 9-ODA, HVA, and 9-HDA. Each oocyte is also challenged with QMP prepared such that the concentration of 9-ODA is ≈100 μM. (B Left) An oocyte injected with RNA encoding AmOr11 fails to respond to 100 μM 9-ODA. (Right) A different oocyte expressing AmOr11 + AmOr2 responds to 100 μM 9-ODA. All applications were 25 s and are indicated by arrowheads (A) or bars (B). AmOr11 + AmOr2 also appeared to respond to a high concentration (100 μM) of 9-HDA (A). However, 9-HDA is synthesized from 9-ODA, and a slight contamination of 9-HDA with 9-ODA is possible.

Fig. 5.

Characterization of the 9-ODA receptor, AmOr11 + AmOr2. (A) An oocyte expressing AmOr11 + AmOr2 is challenged with a range of 9-ODA concentrations. (B) Dose–response relationship for 9-ODA activation of AmOr11 + AmOr2 (EC₅₀ = 280 ± 31 nM, mean ± SEM, n = 10). (C) An oocyte expressing AmOr11 + AmOr2 responds to 100 μM 9-ODA but not to 100 μM methyl oleate (MO), linolenic acid (LA), coniferyl alcohol (CA), or 1-hexadecanol (HD). (D) An oocyte expressing AmOr11 + AmOr2 responds to 100 μM 9-ODA but not to 100 μM hexanol (HEX), linalool (LIN), geraniol (GER), or citral (CIT). All applications were 25 s and are indicated by arrowheads (A, C, and D).