Cineole

Stock solutions of phytochemicals were prepared as 10 mg ml^–1 concentration and stored at 4^oC for further use. Myrtenol, α-bisabolol, carvacrol, phytic acid and ethyl linoleate were procured from Sigma-Aldrich, India. Cineole, theophyline, borneol, oleic acid and α-pinene were procured from Alfa Aeser, India. MEthanol (Sigma-Aldrich, India) was used to dissolve Myrtenol, ethyl linoleate and theophyline. Ethanol (Sigma-Aldrich, India) was used to dissolve α-bisabolol, phytic acid, Cineole, theophyline, borneol, oleic acid and α-pinene.

After euthanizing mice with CO₂, their olfactory turbinates (at the age of P30–P35) were exposed for EOG, which was recorded from multiple turbinates using a MultiClamp 700A amplifier controlled by pClamp software (Molecular Devices). Electrodes were made from standard glass micropipettes filled with 0.5% SeaPlaque agarose (Sigma-Aldrich) in 1× PBS. All odorants, including AA and cineole (Sigma-Aldrich), were diluted in DMSO (Sigma-Aldrich) and mixed to the final working concentration (as shown in Figure 3B) in ultrapure water. Then odorants were delivered in vapor phase along with the humidified airflow to the surface of the tissue. Tissues were allowed 1 minute between subsequent odor deliveries to reduce the adaptation of the EOG response to the previous odorant. The data were analyzed with Clampfit (Molecular Devices).

Odorant responses were elicited by a mixture of Acetophenone (Sigma Aldrich) and Cineole (Sigma Aldrich) diluted to 50 mM in distilled water. The vapor of odorant solution was puffed to the olfactory epithelium by releasing a flow of purified Nitrogen at 20 psi through an odorant bottle connected to an air delivery tube placed at 1 cm from the olfactory epithelium. The puff duration was regulated by a Pneumatic Picopump PV 820 (World Precision Instruments - WPI) triggered by a S48 square pulse electrical stimulator (Grass Technologies) controlled by software using a custom made protocol developed in pCLAMP 10 (Axon Instruments). Odorant responses were elicited by paired-pulses of 100 ms separated by ISIs of 1, 2, 4, 6, 8, 10 and 15 s. We performed 3 trials per condition separated by 1 min intervals.

In the Reserva and Guinea sites, male euglossine bees were sampled for a period of 11 months from June 2006 through May 2007 (except April 2007). In Rio Claro, sampling was conducted from October 2007 to September 2008. At each site, we sampled bees at a single location once every month for 5 h between 08:00 AM and 1:00 PM. We used five chemical baits: cineole (CIN), 1,4-dimethoxybenzene (DMB), methyl cinnamate (MC), methyl salicylate (MS), and vainillin (VAI) (Sigma-Aldrich, St. Louis, MO). We applied each chemical to a small square (5 × 5 cm) of absorbent paper clipped to a tree trunk at a height of 1.5 m.
Baits were placed along an existing trail 10 m away from each other, along mountain ridges. CIN and VAI were replenished every 1.5 h, and the remaining baits were replenished every 2.5 h. Censuses were conducted only during sunny days or slightly cloudy days to avoid short-term variability between samplings rounds. We sequentially collected bees that visited each of the five chemical baits using entomological nets and placed bees in 95% ethanol. Specimens were pinned and deposited at the Museo de Historia Natural of the Universidad de Los Andes (Bogota, Colombia). Species identification was carried out using reference collections of regional euglossine bees as well as taxonomic keys (Bonilla-Gómez and Nates-Parra 1992 ; Roubik and Hanson 2004 ).

AstraZeneca Pharmaceuticals, based in Cairo, Egypt, generously supplied candesartan for this study. We ordered our cholesterol, phospholipids, and cineole from Sigma-Aldrich (St. Louis, MO, USA). We ordered our methanol, ethanol, and chloroform from Cornell Lab (Cairo, Egypt).