Animals. Forager honeybees (A. mellifera carnica) were collected at 2 p.m. at the hive entrance or in an indoor flight room. The bees were immobilized by cooling and mounted in plastic tubes. At 4 p.m., the bees were fed to saturation, and at 4 p.m. on the following day, with 16 μl of SUC solution. All the SUC solutions mentioned in the context of the bee experiments refer to a concentration of 30% (w/v), unless stated otherwise. The animals were kept in a dark humidified chamber overnight at 20° to 24°C.

Learning experiments and Rhodiola feeding in bees. Odor-sugar associative learning experiments were conducted on harnessed bees, as previously described (67). All experiments were performed in the morning (10 to 12 a.m.). The animals were placed next to the training site 30 min before the experiment. Experiments consisted of a five-trial (Fig. 4A) or a three-trial (Fig. 4, D to F, and fig. S6) classical conditioning phase, followed by a single-trial (Fig. 4, D to F) or a three-trial (fig. S6) odor-only presentation test phase. A training trial consisted of the bee being placed in front of an exhaust, followed after 10 s by the presentation of the odor for 5 s and then 3 s later by the presentation of SUC solution to the antennae and the proboscis lasting for 4 s (i.e., an overlap of 2 s between odor and SUC). An extension of the proboscis to the odor was considered to be a response (PER). The total duration of a trial was 30 s; the time between two trials was 10 min. The odor was presented manually with a 20-ml syringe containing a filter paper (1 cm in diameter) with 4 μl of either 1-hexanol (CAS no. 111-27-3) or 1-nonanol (CAS no. 143-08-8) (both 98%; Sigma-Aldrich, Munich, Germany) for each bee.

Bees were fed 16 μl of SUC solution, of which 8 μl contained 0.1, 1, or 10% (w/v) Rhodiola4E root extract. The feeding took place 19 hours before training (1% extract: Fig. 4A), 5 hours after training (i.e., 19 hours before the test; Fig. 4, D and E, and fig. S6), or 29 hours after training (i.e., 19 hours before the test; Fig. 4F). In the control group, the bees were fed 16 μl of SUC solution at these respective time points.

Sugar response experiments in experimentally naïve bees. Bees were fed with 16 μl of SUC solution, of which 8 μl contained 0.1, 1, or 10% (w/v) Rhodiola4E root extract; in the control group, the bees were fed 16 μl of SUC solution. On the following day, the PERs of treated and control bees were noted to concentrations of SUC solution presented to the antenna in an ascending order [0, 0.1, 0.3, 1, 3, 10, and 30% (w/v), according to (68)]. The SUC response score was calculated for each bee by summing the number of PERs it had shown.

Odor response experiments in experimentally naïve bees. Bees were fed 16 μl of SUC solution, of which 8 μl contained 1% (w/v) Rhodiola4E root extract; in the control group, the bees were fed 16 μl of SUC solution. On the following day, the bees were tested for their PER in response to odor (4 μl of either 1-hexanol or 1-nonanol for each bee).

Preparation of Rhodiola4E. To produce Rhodiola4E, dried Rhodiola4 roots were ground in a commercial coffee mill for 3 min and subsequently in a porcelain mortar. Ground roots (100 mg) were stirred for 18 hours at room temperature in 10 ml of ethanol (99%, undenatured) in a light-protected glass bottle. After solvent evaporation, the remaining material was added to SUC solution, vortexed, and heated to 90°C for 60 s.

Stock solutions were kept in darkness at 4°C for up to 7 days. The SUC solution for the control groups was prepared and treated in parallel.

Statistical analyses of behavioral experiments on bees. Included in the analysis were bees that fulfilled two criteria: (i) showing a PER to the SUC solution throughout training and (ii) showing a PER upon SUC stimulation at the very end of the respective experiments. rmANOVA was used with Fisher’s LSD as a post hoc analysis (Statistica version 8.0, StatSoft Inc., Tulsa, OK, USA). We used R version 3.4.1 (69) and lme4 (70) to perform a logistic mixed-effects analysis of the relationship between Rhodiola treatment and the number of PERs in honeybees. To model several trials, we added as fixed effects the time points of testing and the different treatment groups (without an interaction term) to the model. As random effects, we included an intercept for subjects. To model only the test trials, we entered the different treatment groups as fixed effects into the model. As random effects, we had an intercept for subjects. To model the test trials in the concentration dependency experiment, we entered the different treatment groups as fixed effects into the model and added the last trial of the acquisition as a fixed effect. As random effects, we had an intercept for subjects. As optimizers for the model, we used BOBYQA, nloptwrap, and R’s standard. No obvious deviations from homoscedasticity or normality were found by visual inspection of residual plots. We obtained all P values by likelihood ratio tests of the full model with treatment groups against the model without groups as a fixed effect. In Fig. 4 (B to F), bars and error bars represent means and confidence intervals with confidence levels of 95%.

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