Pheromones modulate reward responsiveness and non-associative learning in honey bees

Abstract

Pheromones are chemical messengers that trigger stereotyped behaviors and/or physiological processes in individuals of the same species. Recent reports suggest that pheromones can modulate behaviors not directly related to the pheromonal message itself and contribute, in this way, to behavioral plasticity. We tested this hypothesis by studying the effect of pheromones on sucrose responsiveness and habituation in honey bees. We exposed workers to three pheromone components: geraniol, which in nature is used in an appetitive context, and isopentyl acetate (IPA) and 2-heptanone (2H), which signal aversive situations. Pheromones associated with an aversive context induced a significant decrease of sucrose responsiveness as 40% and 60% of bees exposed to IPA and 2H, respectively, did not respond to any sucrose concentration. In bees that responded to sucrose, geraniol enhanced sucrose responsiveness while 2H, but not IPA, had the opposite effect. Geraniol and IPA had no effect on habituation while 2H induced faster habituation than controls. Overall, our results demonstrate that pheromones modulate reward responsiveness and to a lower degree habituation. Through their effect on sucrose responsiveness they could also affect appetitive associative learning. Thus, besides conveying stereotyped messages, pheromones may contribute to individual and colony-level plasticity by modulating motivational state and learning performances.

Figure 1

Pheromones affect sucrose responsiveness. Proportions of bees that failed to respond to any of the tested sucrose concentrations (including an additional sucrose concentration of 50% w/w delivered at the end of the stimulation sequence) (non-responding bees) following exposure to mineral oil or to one of the three pheromone components (geraniol, IPA and 2H). *p < 0.01; **p < 0.0001.

Figure 2

Pheromone exposure affects sucrose responsiveness. Cumulative proportions of bees showing PER when presented with the six sucrose solutions of increasing concentration (0.1%, 0.3%, 1%, 3%, 10% and 30% w/w). Bees exposed to geraniol (n = 252) and 2H (n = 61) were respectively more and less responsive than control bees exposed to mineral oil (n = 254), (GLMM, geraniol vs oil: p = 0.002; 2H vs oil: p < 0.001). IPA (n = 174) had no significant effect on sucrose responsiveness (IPA vs oil: p = 0.46).

Figure 3

Pheromone exposure affects individual sucrose response scores (SRS). Median, quartiles and max and min (upper and lower whiskers) SRS values of bees exposed to either mineral oil or to one of the three pheromone components (geraniol, IPA and 2H). Red dots represent individual bees. For each bee, SRS was established by measuring PER to a series of six sucrose solutions of increasing concentration (0.1%, 0.3%, 1%, 3%, 10%, and 30% w/w). SRS values ranged between six (bees responding to all 6 concentrations) and 0 (bees not responding to any concentration). Non-responding bees (i.e. bees not responding even to an additional concentration of 50%; see Fig. 1) were excluded from this analysis as their SRS value could not be established. *p = 0.045; **p < 0.0001.

Figure 4

Sucrose response scores are similar following repeated exposures. Median, quartiles and max and min (upper and lower whiskers) SRS values of bees evaluated after a first and second exposure to mineral oil or to one of the three pheromone components (geraniol, IPA and 2H). Red dots represent individual bees. For each bee, SRS was established by measuring PER to a series of six sucrose solutions of increasing concentration (from 0.1% to 30%). SRS values ranged between six (bees responding to all 6 concentrations) and 0 (bees not responding to any concentration). For each of the two sessions, non-responding bees (i.e. bees not responding even to an additional concentration of 50%) were discarded as their SRS value could not be established. In all groups, the SRS remained invariable between the two successive exposures (Wilcoxon test, oil: n = 84, p = 0.18; geraniol: n = 70, p = 0.96; IPA: n = 55, p = 0.98. 2H: n = 32, p = 0.27).

Figure 5

Pheromone exposure affects habituation to sucrose responses. Habituation curves of bees previously exposed to mineral oil (n = 123) or to the three pheromone treatments (geraniol: n = 107, IPA: n = 78, 2H: n = 86). Habituation was measured during 30 consecutive antennal stimulations with 10% sucrose solution. Ten seconds after the last habituation trial, bees were stimulated on the antennae with a 50% sucrose stimulation (Dishabituating Trial or DT) to induce dishabituation. Ten second after the DT, bees were stimulated with the original stimulus used during the training (i.e. 10% sucrose solution) to check for typical response recovery following dishabituation. 2H induced significantly more habituation than mineral oil (GLMM, n = 86, p = 0.0003). Neither geraniol nor IPA affected habituation (GLMM, geraniol: n = 107, p = 0.61; IPA: n = 78, p = 0.77). No significant differences in dishabituation according to treatments were observed. The DT as well as re-stimulating with the original dishabituating stimulus induced a significant response recovery, which did not differ between treatments. This recovery demonstrates that the observed decrease in PER to the 10% sucrose solution was a real case of habituation and was not due to sensory adaptation or fatigue.

Figure 6

Pheromone exposure affects habituation to sucrose responses. Median, quartiles and max and min (upper and lower whiskers) SRS values of habituation scores (see Materials and Methods) for the groups exposed to mineral oil or to the different pheromone components (geraniol, IPA and 2H). Red dots represent individual bees. Bees with a score of 30 responded to all the 30 sucrose stimulations, i.e. did not show any habituation. Non-responding bees (score of 0, 8.2% of those exposed to oil, 13.0% to geraniol, 48.0% to IPA and 48.8% to 2H) were removed from the analysis as habituation cannot be assessed in this case. **p = 0.0003.