Caffeine in floral nectar enhances a pollinator's memory of reward

Abstract

Plant defense compounds occur in floral nectar, but their ecological role is not well understood. We provide evidence that plant compounds pharmacologically alter pollinator behavior by enhancing their memory of reward. Honeybees rewarded with caffeine, which occurs naturally in nectar of Coffea and Citrus species, were three times as likely to remember a learned floral scent as were honeybees rewarded with sucrose alone. Caffeine potentiated responses of mushroom body neurons involved in olfactory learning and memory by acting as an adenosine receptor antagonist. Caffeine concentrations in nectar did not exceed the bees' bitter taste threshold, implying that pollinators impose selection for nectar that is pharmacologically active but not repellent. By using a drug to enhance memories of reward, plants secure pollinator fidelity and improve reproductive success.

Figure 1

(A) Caffeine concentration in Coffea and Citrus sp. and a cup of instant coffee. Caffeine concentration depended on species within each genus (Coffea: Kruskal-Wallis, χ₂² = 28.1, P < 0.001; Citrus: Kruskal-Wallis, χ₂² = 6.98, P = 0.030); C. canephora had the highest mean concentration of all species sampled. (B) The sum of the concentration of sucrose, glucose and fructose (total nectar sugars) depended on species (1-way ANOVA: F_{5, 161} = 4.64, P < 0.001) and was greatest in Citrus maxima and hybrids (citron, lemons, clementines). (C. can. = Coffea canephora, N = 34; C. lib. = Coffea liberica, N = 31; C. arab. = Coffea arabica, N = 27; C. par. = Citrus paradisi and hybrids, N_cp = 17; C. max. = Citrus maxima and hybrids, N = 5; C. sin. and C. ret. = Citrus sinensis and Citrus reticulata, N_CS = 7, N_CR = 5 – data for these two species was pooled).

Figure 2

(A) The rate of learning of bees conditioned with an odour stimulus paired with a 0.7 M sucrose reward containing caffeine. The rate of learning was slightly greater for the bees fed caffeine in reward during conditioning (logistic regression, χ₁² = 4.85, P = 0.028). N ≥ 79 for all groups. (B) Memory recall test for odours at 10 min (clear bars) or 24 h (red bars) after bees had been trained as in (A). Bright red bars indicate that the response at 24 h was significantly different from the control (0.7 M sucrose) (least-squares contrasts: P < 0.05); dark red bars were not significantly different. Nectar-levels of caffeine are indicated by hatching. N > 79 for each group. (C) Bees fed 0.1 mM caffeine in sucrose (red bars) were more likely to remember the conditioned odour sucrose alone (white bars) (logistic regression, χ₁² = 9.04, P < 0.003) at 24 h and 72 h after conditioning. N = 40 per group.

Figure 3

The effect of caffeine on Kenyon cells. (A, B) Example traces from a KC in intact honeybee brain recorded under voltage-clamp (A, V_H = −73 mV) and current-clamp (B; at resting V_M), showing the increase in I_M and depolarization evoked by bath application of caffeine (100 μM), and subsequent reversal by the nAChR antagonist d-TC (500 μM). (C, D) Mean data showing the reversal by d-TC (500 μM) of the effect of caffeine (Caff; 100 μM) on I_M (C; N = 6, t₅ = 4.03, P = 0.010; t₅ = 4.07, P = 0.010) and V_M (D; N = 6, t₅ = 34.1, P < 0.001; t₅ = 12.0, P < 0.001). (E, F) Comparison of the mean effects of caffeine and DPCPX on I_M (E, Caff: N = 10, t₉ = 3.84, P = 0.004; DPCPX: N = 6, t₅ = 4.04, P = 0.010) and V_M (F, Caff: N = 6, t₅ = 34.1, P < 0.001; DPCPX: N = 6, t₅ = 3.39, P = 0.019). (G, H) Example traces (G; rising phase shown on an expanded time-scale below) and mean data (H, Rate of rise: N = 6, t₅ = 2.20, P = 0.079; τ_decay: N = 9, t₈ = 3.54, P = 0.008) showing that DPCPX (100 nM) and caffeine (100 μM) slowed the decay and, in 6 of 9 KCs, potentiated the fast component of the response evoked by exogenous ACh. (Student’s paired t-test used in all comparisons).

Figure 4

Bees are more likely to reject sucrose solutions containing caffeine of concentrations greater than 1 mM (logistic regression, χ₄² = 23.4, P < 0.001; for 0.7M and 1.0M, 1 mM caffeine vs sucrose post hoc, P < 0.05; for 0.3M, 100 mM caffeine vs sucrose post hoc, P < 0.05). Bees were less likely to drink 0.3 M sucrose (pale pink diamonds) than 0.7M (pink circles) or 1.0M solutions (red triangles) (logistic regression, χ₂² = 8.69, P = 0.013). Mean responses + SE. N_0.3M = 29, N_0.7M = 100, N_1.0M = 20.