Plant defence compounds occur in floral nectar, but their ecological role is not well-understood. We provide the first evidence that plant compounds pharmacologically alter pollinator behaviour by enhancing their memory of reward. Honeybees rewarded with caffeine, which occurs naturally in nectar of Coffea and Citrus species, were three times more likely to remember a learned floral scent than those 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 never exceeded 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.Many drugs commonly consumed by humans are produced by plants as a form of toxic defence against herbivores (1, 2). While plant-derived drugs like caffeine or nicotine are lethal in high doses (3-5), they have pharmacological effects at low doses that affect mammalian behaviour. For example, low doses of caffeine are mildly rewarding and enhance cognitive performance and memory retention (6). Interestingly, caffeine has been detected in low doses in the floral nectar and pollen of Citrus (7), but whether it has an ecological function is unknown.Two caffeine-producing plant genera, Citrus and Coffea, have large floral displays with strong scents and produce more fruits and seeds when pollinated by bees (8, 9). If caffeine confers a selective advantage when these plants interact with pollinators, we might expect it to be commonly encountered in nectar. We measured caffeine in the nectar of 3 species of Coffea (C. canephora, C. arabica, and C. liberica) and 4 species of Citrus (C. paradisi, C. maxima, C. sinesis, C. reticulata) using liquid chromatography-mass spectrometry (10, Fig. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts S1A). When caffeine was present, its concentration ranged from 0.003 -0.253 mM. The median caffeine concentration in both genera was not significantly different (Fig. 1A, MannWhitney, Z = −1.09, P = 0.272). Caffeine was more common in the nectar of C. canephora than in C. arabica or C. liberica (Coffea: logistic regression χ 2 2 = 11.1, P = 0.004); it was always present in Citrus nectar. The mean total nectar sugar concentration ranged from 0.338-0.843 M (Fig. 1B, see Fig S1B for individual sugars). Caffeine concentration in nectar did not correlate with total sugar concentration (Pearson's r = 0.063, P = 0.596).We hypothesized that caffeine could affect the learning and memory of foraging pollinators.To test this, we trained individual honeybees to associate floral scent with 0.7 M sucrose and 7 different concentrations of caffeine and tested their olfactory memory. Using a method for classical conditioning of feeding responses (proboscis extension reflex, 11), bees...

Summary1. The ecological function of secondary metabolites in plant defence against herbivores is well established, but their role in plant-pollinator interactions is less obvious. Nectar is the major reward for pollinators, so the occurrence of defence chemicals in the nectar of many species is unexpected. However, increasing evidence supports a variety of potential benefits for both plant and pollinator from these compounds. 2. Beneficial effects may include: (i) mediating specialization in plant-pollinator interactions, (ii) protecting nectar from robbery or larceny and (iii) microbial activity including preservation of nutrients in nectar from degradation and reduction in disease levels in pollinators. 3. Secondary metabolites in nectar can be toxic or repellent to flower visitors, but equally they can go undetected or even make nectar more apparent or attractive. These biological effects are concentration dependent, so must be considered at a range of ecologically relevant doses. For example, caffeine occurs in nectar and improves honeybee memory for odours associated with food rewards, which enhances pollen transfer at naturally occurring concentrations but is repellent to honeybees at higher concentrations. 4. This review synthesizes evidence from recent literature that supports selection for secondary metabolites in floral nectar as an adaptation that drives the co-evolution between plants and their pollinators. However, their presence in nectar could still simply be a consequence of their defensive role elsewhere in the plant (pleiotropy). We highlight the need for more studies demonstrating measurable benefits to the plant, the importance of exposure levels and effects on target species beyond the current emphasis on alkaloids and bees.