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Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors
Report
Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors Highlights d
Caffeinated forage causes honeybees to overestimate forage quality
d
Caffeine leads to individual- and colony-level effects in foraging and recruitment
d
A model demonstrates how food stores could decrease from caffeine-enhanced foraging
d
Caffeine in nectar may ultimately benefit the plant, not the pollinator
Couvillon et al., 2015, Current Biology 25, 1–4 November 2, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2015.08.052
Authors Margaret J. Couvillon, Hasan Al Toufailia, Thomas M. Butterfield, Felix Schrell, Francis L.W. Ratnieks, Roger Schu¨rch
Correspondence [email protected]
In Brief Couvillon et al. show that naturally occurring caffeinated forage tricks honeybees into acting as if nectar quality, measured by bees as sugar content, is higher than it really is. Workers increase foraging and recruitment behaviors, which ultimately quadruples colony-level recruitment, tempting the colony into sub-optimal foraging strategies.
Please cite this article in press as: Couvillon et al., Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.08.052
Current Biology
Report Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors Margaret J. Couvillon,1,* Hasan Al Toufailia,1 Thomas M. Butterfield,2 Felix Schrell,1 Francis L.W. Ratnieks,1 and Roger Schu¨rch1,3 1Laboratory
of Apiculture and Social Insects (LASI), School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK 3Clinical Trials Unit (CTU), University of Bern, 3012 Bern, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.08.052 2School
SUMMARY
In pollination, plants provide food reward to pollinators who in turn enhance plant reproduction by transferring pollen, making the relationship largely cooperative; however, because the interests of plants and pollinators do not always align, there exists the potential for conflict, where it may benefit both to cheat the other [1, 2]. Plants may even resort to chemistry: caffeine, a naturally occurring, bitter-tasting, pharmacologically active secondary compound whose main purpose is to detract herbivores [3–6], is also found in lower concentrations in the nectar of up to 55% of plants, even though nectar, unlike leaves, is made to be consumed by pollinators [5, 7, 8]. A recent laboratory study showed that caffeine may lead to efficient and effective foraging by aiding honeybee memory of a learned olfactory association [4], suggesting that caffeine may enhance bee reward perception. However, without field data, the wider ecological significance of caffeinated nectar remains difficult to interpret. Here we demonstrate in the field that caffeine generates significant individual- and colony-level effects in free-flying worker honeybees. Compared to a control, a sucrose solution with field-realistic doses of caffeine caused honeybees to significantly increase their foraging frequency, waggle dancing probability and frequency, and persistency and specificity to the forage location, resulting in a quadrupling of colony-level recruitment. An agent-based model also demonstrates how caffeine-enhanced foraging may reduce honey storage. Overall, caffeine causes bees to overestimate forage quality, tempting the colony into sub-optimal foraging strategies, which makes the relationship between pollinator and plant less mutualistic and more exploitative. RESULTS AND DISCUSSION Caffeine Increases Individual Honeybee Foraging Frequency We trained (day 0) individually marked honeybees (15 per hive per treatment, three hives in total) to forage at one of two feeders
190 m from the observation hive using standard procedures (see Supplemental Experimental Procedures) [9–11]. For 3 hr, the foragers collected 1 M unscented sucrose solution, either without (control) or with (treatment) caffeine at a concentration found naturally in nectar (25 ppm, Sigma Aldrich Reagent Powder; see Supplemental Experimental Procedures) [4, 5]. Concurrently, we quantified three foraging and recruitment behaviors (foraging frequency, waggle dance propensity, and waggle dance frequency; see each section below for definitions). Previous research has shown that an increase in these behaviors directly correlates with an increase in sucrose solution molarity (e.g., reward quality), to which the honeybees are exceptionally sensitive [9, 12–16]. In other words, any behavioral differences observed between treated versus untreated bees would indicate that they perceived the reward differently, even though they were identical in molarity. As predicted, we found that foraging frequency, or the number of foraging visits that individual bees made across the standardized 3 hr experimental period, increased significantly when bees collected sucrose solution with caffeine compared to without (median visits per 3 hr: caffeine 23.5 versus control 18.5, Mann-Whitney, W = 1040, p = 0.048; Figure 1). Caffeine also decreased inter-bee variation in foraging frequency (first quartile for caffeine versus control, 17 versus 7; Figure 1), meaning that under the influence of caffeine, there are fewer bees foraging at the lower frequencies. Caffeine Increases Individual Bee Waggle Dancing Propensity and Frequency, which Combine to Quadruple Colony-Level Recruitment Simultaneously, we monitored waggle dances made by the marked foragers returning to the observation hive (see Supplemental Experimental Procedures). The waggle dance is a unique recruitment behavior in which a returning honeybee that foraged profitably communicates to nestmates the distance and direction from the hive to that forage [9, 17]. A worker making repeated visits to the forage location will modulate whether or not she dances at all (dance propensity) and, once she is dancing, how often she dances (dance frequency) based on sucrose solution concentration [9, 12]. As predicted, we found a strong effect of treatment on recruitment behavior. Over the 3 hr, a significantly greater proportion of bees foraging at the caffeine feeder performed a dance (dance propensity with caffeine: 88% or 42/48) compared to the bees at the control feeder (dance propensity with control: 67% or 39/58; binomial generalized linear mixed model [GLMM] on proportion of
Current Biology 25, 1–4, November 2, 2015 ª2015 Elsevier Ltd All rights reserved 1
Please cite this article in press as: Couvillon et al., Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.08.052
Figure 1. Caffeine Increases Honeybee Foraging Frequency Bees foraging at a feeder with 1 M sucrose solution with caffeine made significantly more visits (median visits per 3 hr: 23.5, black) compared to bees visiting a feeder with equal molarity solution without caffeine (18.5, gray). Whiskers extend to 1.53 interquartile range or extreme values.
dancing/non-dancing bees with a random intercept for hives; treatment effect: likelihood ratio test [LRT] = 6.27, df = 1, p = 0.012; Figure 2A). Once a bee began to dance, caffeine more than tripled the dance frequency (median number of dances per bee per 3 hr for caffeine versus control = 9.5 versus 3; Poisson GLMM on number of dances per bee per treatment with random intercepts for hives, treatment effect: LRT = 58.36, df = 1, p = 2.19e 14; Figure 2B). How do these individual behaviors impact the colony? Based on these data, we calculated that under the influence of caffeine, a colony will experience an overall quadrupling in total number of recruitment events (dances) compared to an equal-sized colony foraging on equal-molarity control: if colonies possess n foragers, the caffeine colony will experience 8.36 3 n dances versus the control colony of 2.01 3 n dances (caffeine: 88% of n foragers dancing 3 9.5 dances per bee / 3 hr = 8.36n; control: 67% of n foragers dancing 3 3 dances per bee / 3 hr = 2.01n; Figure 2C). At the level of the colony, recruitment of new foragers is linearly related to the amount of dancing [12, 13]. Therefore, a colony exploiting a resource with caffeine will send out approximately four times as many new recruits per unit of time compared to the control, a difference that will compound with the positive feedback of recruitment. Caffeine Increases Persistency and Site Specificity We then wished to assess the post-exposure effects of caffeine on honeybee foragers. On the morning of day 1, we placed two empty feeders where the caffeine and control feeders were on day 0, and we monitored both for marked forager visits (see Supplemental Experimental Procedures). Persistency, or the
tendency of honeybees to return to a feeding location after it has become unrewarding, is strongly affected by sucrose concentration, where foragers are more persistent for locations where they experienced sweeter forage [15]. As predicted, bees that had received caffeine on day 0 returned more persistently on days 1–6 to the unrewarding feeders, both for more days and more often within days: caffeine and the number of visits during the 3 hr experiment on day 0 (i.e., access duration; see Supplemental Experimental Procedures) significantly interact to predict the number of days that a bee will persistently return (n = 104, Poisson GLMM days persistent against fixed factors of treatment, access duration, and an interaction; treatment 3 access duration: df = 1, LRT = 5.8541, p = 0.016). The interaction is that a bee with only little access duration will be more persistent if she was exposed to caffeine. Additionally, the number of persistent visits per day depends on both the access duration and treatment: visits per day declined over time in both treatments equally (Poisson GLMM number of visits per day against fixed factors of caffeine treatment and time and access duration; time: df = 1, LRT = 337.41, p < 0.001; experimental visits: df = 1, LRT = 11.85, p < 0.001; treatment: df = 1, LRT: df = 1, LRT = 4.47, p = 0.034; Figure 3A). In other words, persistency declines equally for both, but caffeinetreated bees are 23% more persistent on day 1, shifting the decay upward (Figure 3A). Concurrently, we monitored site specificity, which is the disinclination that a persistent forager will investigate the other feeder 2 m away (see Supplemental Experimental Procedures), to which she was not trained on day 0. Such searching behavior (low site specificity) is normally considered adaptive, as a rewarding area is likely to contain similar reward nearby. We found that caffeine significantly increased site specificity: the proportion of bees that investigated only their feeder was significantly higher for caffeine versus control (58%, n = 45, versus 26%, n = 42; binomial GLMM proportion exploring both feeders against caffeine treatment versus null of no treatment, hive as random factor, LRT = 9.06, df = 1, p = 0.003; Figure 3B). In other words, under the influence of caffeine, foragers are less likely to explore the area for alternative forage. An Environment with Caffeinated Forage May Be Modeled to Demonstrate a Daily 14.5% Decrease in Honey Production Bees feeding on caffeine-containing nectar of course still receive a reward; however, as demonstrated by these experimental effects, the bees may be foraging sub-optimally because they overvalue a caffeinated nectar source compared to a decaffeinated source with equal energy. Plants could potentially lower their nectar investment (sucrose content) but still receive high visitation if caffeine is present. We explored this idea further by modifying, based on our data, an agent-based model [18]. Patches were designated as ‘‘caffeine’’ or ‘‘control.’’ If an agent visited a caffeine patch, it experienced an increased likelihood to recruit for it and to revisit it the next day. These modifiers were chosen so that a control probability to dance of 67% increased to 88% (see Figure 2A). Simultaneously, the plants lowered their nectar sugar concentration from 1.0 mol/l to 0.8 mol/l (see Supplemental Experimental Procedures), while we monitored colony
2 Current Biology 25, 1–4, November 2, 2015 ª2015 Elsevier Ltd All rights reserved
Please cite this article in press as: Couvillon et al., Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.08.052
Figure 2. Caffeine Increases Individual Bee Waggle Dancing Propensity and Frequency, which Combine to Quadruple Colony-Level Recruitment (A) A significantly higher proportion of honeybees foraging on sucrose solution with caffeine performed waggle dances (88%, black) compared to control (67%, gray). (B) Caffeine significantly tripled the median dancing frequency per 3 hr (9.5, black) compared to control (3, gray). (C) The individual effects in (A) and (B) combine to generate a colony-level quadrupling in the number of dances experienced in caffeine (black) versus control (gray) colonies for equal-molarity forage. Error bars represent SEM.
weight (kg honey stores). The results support the prediction such that with 40% caffeine patches in the environment, approximately 14.5% less honey will be produced each day compared to production by colonies foraging in a caffeine-free environment (Figure 4). Although evolutionary innovation often arises from genetic variation, new adaptations may additionally occur as a byproduct of unrelated processes, e.g., cooption or exaptation [19]. Caffeine occurs naturally in the leaves and seeds of many genera, including Citrus, Coffea, and Camellia, and possesses
a bitter taste that reduces palatability/digestibility to herbivores, thus acting as a chemical defense [3, 6, 7, 20]. The making and mobilization of these secondary compounds may have resulted in their accidental co-occurrence in nectar, where they may have subsequently been coopted for an additional role in plant reproduction [4, 5, 8, 21]. Nicotine, another secondary compound, has been shown to increase plant reproductive fitness by slightly decreasing nectar palatability to pollinators, who then move more quickly between flowers [22]. In this way, the effect of nicotine in nectar on pollinators mirrors its intended role in chemical
Figure 3. Caffeine Increases Persistency and Site Specificity (A) Initial persistency (number of visits per day to the now unrewarding feeder) significantly increased with caffeine, where bees receiving caffeine during training on day 0 (black line) visited 23% more compared to control (gray line) on day 1 of the feeder becoming unrewarding. Visits per day declined equally over time in both treatments. Dot size corresponds to sample size. (B) Caffeine significantly increases site specificity, such that a greater proportion of bees exposed to caffeine (58%, black) persistently returned to investigate only the unrewarding feeder to which they were initially trained compared to control bees (26%, gray), who were more likely to return and investigate both unrewarding feeders. Error bars represent SEM.
Current Biology 25, 1–4, November 2, 2015 ª2015 Elsevier Ltd All rights reserved 3
Please cite this article in press as: Couvillon et al., Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.08.052
Received: July 28, 2015 Revised: August 19, 2015 Accepted: August 24, 2015 Published: October 15, 2015 REFERENCES 1. Irwin, R.E., Bronstein, J.L., Manson, J.S., and Richardson, L. (2010). Nectar robbing: ecological and evolutionary perspectives. Annu. Rev. Ecol. Evol. Syst. 41, 271–292. 2. Dafni, A. (1984). Mimicry and deception in pollination. Annu. Rev. Ecol. Syst. 15, 259–278. 3. Berenbaum, M., and Rosenthal, G.A. (1992). Herbivores: Their Interactions with Secondary Plant Metabolites (Academic Press). 4. Wright, G.A., Baker, D.D., Palmer, M.J., Stabler, D., Mustard, J.A., Power, E.F., Borland, A.M., and Stevenson, P.C. (2013). Caffeine in floral nectar enhances a pollinator’s memory of reward. Science 339, 1202–1204. 5. Singaravelan, N., Nee’man, G., Inbar, M., and Izhaki, I. (2005). Feeding responses of free-flying honeybees to secondary compounds mimicking floral nectars. J. Chem. Ecol. 31, 2791–2804. 6. Kretschmar, J.A., and Baumann, T.W. (1999). Caffeine in Citrus flowers. Phytochemistry 52, 19–23.
Figure 4. Caffeine May Cause a Cost to the Colony Results of an agent-based model indicate that plants with nectar containing caffeine may lower their nectar sugar concentrations without decreasing their attractiveness to pollinators, resulting in colony weight loss. Caffeine, accompanied by a drop in nectar quality (black circle), is predicted to cause colonies to produce approximately 14.5% less honey per day compared both to colonies foraging in an environment with caffeine but without a drop in nectar quality (dark gray circle) and colonies foraging in an environment with no caffeine and no drop in nectar quality (light gray circle). Error bars represent SEM.
defense (e.g., decreasing palatability). In contrast, here we have shown that the effects of caffeine in nectar are akin to drugging, where the pollinator’s perception of the forage quality is altered, which in turn changes its individual behaviors. These behaviors may scale to colony-level effects, as predicted by the model, a hypothesis that could be experimentally tested. As such, these data provide a fascinating example of the ongoing tension between conflict and cooperation in mutualisms, and the diversity and subtlety of the mechanisms that are used to manipulate partners. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.cub. 2015.08.052. AUTHOR CONTRIBUTIONS M.J.C. and R.S. conceived and designed the experiment and oversaw data collection. M.J.C. wrote the manuscript. R.S. performed the statistical analyses. F.L.W.R. contributed valuable discussions and contributed to the writing of the manuscript. H.A.T., T.M.B., and F.S. assisted with data collection. ACKNOWLEDGMENTS M.J.C. is funded by the Nineveh Charitable Trust. T.M.B. is funded by the Natural Environment Research Council (NERC) and the University of Sussex. H.A.T. is funded by Damascus University. R.S. is funded by Rowse Honey Ltd. Additional research funding was provided by Waitrose Ltd. and Burt’s Bees.
7. Mustard, J.A. (2014). The buzz on caffeine in invertebrates: effects on behavior and molecular mechanisms. Cell. Mol. Life Sci. 71, 1375–1382. 8. Adler, L.S. (2000). The ecological significance of toxic nectar. Oikos 91, 409–420. 9. von Frisch, K. (1967). The Dance Language and Orientation of Bees (Harvard University Press). 10. Gru¨ter, C., and Ratnieks, F.L.W. (2011). Honeybee foragers increase the use of waggle dance information when private information becomes unrewarding. Anim. Behav. 81, 949–954. 11. Schu¨rch, R., Couvillon, M.J., Burns, D.D.R., Tasman, K., Waxman, D., and Ratnieks, F.L.W. (2013). Incorporating variability in honey bee waggle dance decoding improves the mapping of communicated resource locations. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 199, 1143–1152. 12. Seeley, T.D. (1995). The Wisdom of the Hive (Harvard University Press). 13. Seeley, T.D. (1997). Honey bee colonies are group-level adaptive units. Am. Nat. 150 (Suppl 1 ), S22–S41. 14. Seeley, T.D. (1994). Honey bee foragers as sensory units of their colonies. Behav. Ecol. Sociobiol. 34, 51–62. 15. Al Toufailia, H., Gru¨ter, C., and Ratnieks, F.L.W. (2013). Persistence to unrewarding feeding locations by honeybee foragers (Apis mellifera): the effects of experience, resource profitability and season. Ethology 119, 1096–1106. 16. Seeley, T.D., Camazine, S., and Sneyd, J. (1991). Collective decision-making in honey bees: how colonies choose among nectar sources. Behav. Ecol. Sociobiol. 28, 277–290. 17. Couvillon, M.J. (2012). The dance legacy of Karl von Frisch. Insectes Soc. 59, 297–306. 18. Schu¨rch, R., and Gru¨ter, C. (2014). Dancing bees improve colony foraging success as long-term benefits outweigh short-term costs. PLoS ONE 9, e104660. 19. Gould, S.J., and Vrba, E.S. (1982). Exaptation—a missing term in the science of form. Paleobiology 8, 4–15. 20. Ashihara, H., and Crozier, A. (2001). Caffeine: a well known but little mentioned compound in plant science. Trends Plant Sci. 6, 407–413. 21. Armbruster, W.S., Lee, J., and Baldwin, B.G. (2009). Macroevolutionary patterns of defense and pollination in Dalechampia vines: adaptation, exaptation, and evolutionary novelty. Proc. Natl. Acad. Sci. USA 106, 18085–18090. 22. Kessler, D., Gase, K., and Baldwin, I.T. (2008). Field experiments with transformed plants reveal the sense of floral scents. Science 321, 1200–1202.
4 Current Biology 25, 1–4, November 2, 2015 ª2015 Elsevier Ltd All rights reserved
Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors Highlights d
Caffeinated forage causes honeybees to overestimate forage quality
d
Caffeine leads to individual- and colony-level effects in foraging and recruitment
d
A model demonstrates how food stores could decrease from caffeine-enhanced foraging
d
Caffeine in nectar may ultimately benefit the plant, not the pollinator
Couvillon et al., 2015, Current Biology 25, 1–4 November 2, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2015.08.052
Authors Margaret J. Couvillon, Hasan Al Toufailia, Thomas M. Butterfield, Felix Schrell, Francis L.W. Ratnieks, Roger Schu¨rch
Correspondence [email protected]
In Brief Couvillon et al. show that naturally occurring caffeinated forage tricks honeybees into acting as if nectar quality, measured by bees as sugar content, is higher than it really is. Workers increase foraging and recruitment behaviors, which ultimately quadruples colony-level recruitment, tempting the colony into sub-optimal foraging strategies.
Please cite this article in press as: Couvillon et al., Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.08.052
Current Biology
Report Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors Margaret J. Couvillon,1,* Hasan Al Toufailia,1 Thomas M. Butterfield,2 Felix Schrell,1 Francis L.W. Ratnieks,1 and Roger Schu¨rch1,3 1Laboratory
of Apiculture and Social Insects (LASI), School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK 3Clinical Trials Unit (CTU), University of Bern, 3012 Bern, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.08.052 2School
SUMMARY
In pollination, plants provide food reward to pollinators who in turn enhance plant reproduction by transferring pollen, making the relationship largely cooperative; however, because the interests of plants and pollinators do not always align, there exists the potential for conflict, where it may benefit both to cheat the other [1, 2]. Plants may even resort to chemistry: caffeine, a naturally occurring, bitter-tasting, pharmacologically active secondary compound whose main purpose is to detract herbivores [3–6], is also found in lower concentrations in the nectar of up to 55% of plants, even though nectar, unlike leaves, is made to be consumed by pollinators [5, 7, 8]. A recent laboratory study showed that caffeine may lead to efficient and effective foraging by aiding honeybee memory of a learned olfactory association [4], suggesting that caffeine may enhance bee reward perception. However, without field data, the wider ecological significance of caffeinated nectar remains difficult to interpret. Here we demonstrate in the field that caffeine generates significant individual- and colony-level effects in free-flying worker honeybees. Compared to a control, a sucrose solution with field-realistic doses of caffeine caused honeybees to significantly increase their foraging frequency, waggle dancing probability and frequency, and persistency and specificity to the forage location, resulting in a quadrupling of colony-level recruitment. An agent-based model also demonstrates how caffeine-enhanced foraging may reduce honey storage. Overall, caffeine causes bees to overestimate forage quality, tempting the colony into sub-optimal foraging strategies, which makes the relationship between pollinator and plant less mutualistic and more exploitative. RESULTS AND DISCUSSION Caffeine Increases Individual Honeybee Foraging Frequency We trained (day 0) individually marked honeybees (15 per hive per treatment, three hives in total) to forage at one of two feeders
190 m from the observation hive using standard procedures (see Supplemental Experimental Procedures) [9–11]. For 3 hr, the foragers collected 1 M unscented sucrose solution, either without (control) or with (treatment) caffeine at a concentration found naturally in nectar (25 ppm, Sigma Aldrich Reagent Powder; see Supplemental Experimental Procedures) [4, 5]. Concurrently, we quantified three foraging and recruitment behaviors (foraging frequency, waggle dance propensity, and waggle dance frequency; see each section below for definitions). Previous research has shown that an increase in these behaviors directly correlates with an increase in sucrose solution molarity (e.g., reward quality), to which the honeybees are exceptionally sensitive [9, 12–16]. In other words, any behavioral differences observed between treated versus untreated bees would indicate that they perceived the reward differently, even though they were identical in molarity. As predicted, we found that foraging frequency, or the number of foraging visits that individual bees made across the standardized 3 hr experimental period, increased significantly when bees collected sucrose solution with caffeine compared to without (median visits per 3 hr: caffeine 23.5 versus control 18.5, Mann-Whitney, W = 1040, p = 0.048; Figure 1). Caffeine also decreased inter-bee variation in foraging frequency (first quartile for caffeine versus control, 17 versus 7; Figure 1), meaning that under the influence of caffeine, there are fewer bees foraging at the lower frequencies. Caffeine Increases Individual Bee Waggle Dancing Propensity and Frequency, which Combine to Quadruple Colony-Level Recruitment Simultaneously, we monitored waggle dances made by the marked foragers returning to the observation hive (see Supplemental Experimental Procedures). The waggle dance is a unique recruitment behavior in which a returning honeybee that foraged profitably communicates to nestmates the distance and direction from the hive to that forage [9, 17]. A worker making repeated visits to the forage location will modulate whether or not she dances at all (dance propensity) and, once she is dancing, how often she dances (dance frequency) based on sucrose solution concentration [9, 12]. As predicted, we found a strong effect of treatment on recruitment behavior. Over the 3 hr, a significantly greater proportion of bees foraging at the caffeine feeder performed a dance (dance propensity with caffeine: 88% or 42/48) compared to the bees at the control feeder (dance propensity with control: 67% or 39/58; binomial generalized linear mixed model [GLMM] on proportion of
Current Biology 25, 1–4, November 2, 2015 ª2015 Elsevier Ltd All rights reserved 1
Please cite this article in press as: Couvillon et al., Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.08.052
Figure 1. Caffeine Increases Honeybee Foraging Frequency Bees foraging at a feeder with 1 M sucrose solution with caffeine made significantly more visits (median visits per 3 hr: 23.5, black) compared to bees visiting a feeder with equal molarity solution without caffeine (18.5, gray). Whiskers extend to 1.53 interquartile range or extreme values.
dancing/non-dancing bees with a random intercept for hives; treatment effect: likelihood ratio test [LRT] = 6.27, df = 1, p = 0.012; Figure 2A). Once a bee began to dance, caffeine more than tripled the dance frequency (median number of dances per bee per 3 hr for caffeine versus control = 9.5 versus 3; Poisson GLMM on number of dances per bee per treatment with random intercepts for hives, treatment effect: LRT = 58.36, df = 1, p = 2.19e 14; Figure 2B). How do these individual behaviors impact the colony? Based on these data, we calculated that under the influence of caffeine, a colony will experience an overall quadrupling in total number of recruitment events (dances) compared to an equal-sized colony foraging on equal-molarity control: if colonies possess n foragers, the caffeine colony will experience 8.36 3 n dances versus the control colony of 2.01 3 n dances (caffeine: 88% of n foragers dancing 3 9.5 dances per bee / 3 hr = 8.36n; control: 67% of n foragers dancing 3 3 dances per bee / 3 hr = 2.01n; Figure 2C). At the level of the colony, recruitment of new foragers is linearly related to the amount of dancing [12, 13]. Therefore, a colony exploiting a resource with caffeine will send out approximately four times as many new recruits per unit of time compared to the control, a difference that will compound with the positive feedback of recruitment. Caffeine Increases Persistency and Site Specificity We then wished to assess the post-exposure effects of caffeine on honeybee foragers. On the morning of day 1, we placed two empty feeders where the caffeine and control feeders were on day 0, and we monitored both for marked forager visits (see Supplemental Experimental Procedures). Persistency, or the
tendency of honeybees to return to a feeding location after it has become unrewarding, is strongly affected by sucrose concentration, where foragers are more persistent for locations where they experienced sweeter forage [15]. As predicted, bees that had received caffeine on day 0 returned more persistently on days 1–6 to the unrewarding feeders, both for more days and more often within days: caffeine and the number of visits during the 3 hr experiment on day 0 (i.e., access duration; see Supplemental Experimental Procedures) significantly interact to predict the number of days that a bee will persistently return (n = 104, Poisson GLMM days persistent against fixed factors of treatment, access duration, and an interaction; treatment 3 access duration: df = 1, LRT = 5.8541, p = 0.016). The interaction is that a bee with only little access duration will be more persistent if she was exposed to caffeine. Additionally, the number of persistent visits per day depends on both the access duration and treatment: visits per day declined over time in both treatments equally (Poisson GLMM number of visits per day against fixed factors of caffeine treatment and time and access duration; time: df = 1, LRT = 337.41, p < 0.001; experimental visits: df = 1, LRT = 11.85, p < 0.001; treatment: df = 1, LRT: df = 1, LRT = 4.47, p = 0.034; Figure 3A). In other words, persistency declines equally for both, but caffeinetreated bees are 23% more persistent on day 1, shifting the decay upward (Figure 3A). Concurrently, we monitored site specificity, which is the disinclination that a persistent forager will investigate the other feeder 2 m away (see Supplemental Experimental Procedures), to which she was not trained on day 0. Such searching behavior (low site specificity) is normally considered adaptive, as a rewarding area is likely to contain similar reward nearby. We found that caffeine significantly increased site specificity: the proportion of bees that investigated only their feeder was significantly higher for caffeine versus control (58%, n = 45, versus 26%, n = 42; binomial GLMM proportion exploring both feeders against caffeine treatment versus null of no treatment, hive as random factor, LRT = 9.06, df = 1, p = 0.003; Figure 3B). In other words, under the influence of caffeine, foragers are less likely to explore the area for alternative forage. An Environment with Caffeinated Forage May Be Modeled to Demonstrate a Daily 14.5% Decrease in Honey Production Bees feeding on caffeine-containing nectar of course still receive a reward; however, as demonstrated by these experimental effects, the bees may be foraging sub-optimally because they overvalue a caffeinated nectar source compared to a decaffeinated source with equal energy. Plants could potentially lower their nectar investment (sucrose content) but still receive high visitation if caffeine is present. We explored this idea further by modifying, based on our data, an agent-based model [18]. Patches were designated as ‘‘caffeine’’ or ‘‘control.’’ If an agent visited a caffeine patch, it experienced an increased likelihood to recruit for it and to revisit it the next day. These modifiers were chosen so that a control probability to dance of 67% increased to 88% (see Figure 2A). Simultaneously, the plants lowered their nectar sugar concentration from 1.0 mol/l to 0.8 mol/l (see Supplemental Experimental Procedures), while we monitored colony
2 Current Biology 25, 1–4, November 2, 2015 ª2015 Elsevier Ltd All rights reserved
Please cite this article in press as: Couvillon et al., Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.08.052
Figure 2. Caffeine Increases Individual Bee Waggle Dancing Propensity and Frequency, which Combine to Quadruple Colony-Level Recruitment (A) A significantly higher proportion of honeybees foraging on sucrose solution with caffeine performed waggle dances (88%, black) compared to control (67%, gray). (B) Caffeine significantly tripled the median dancing frequency per 3 hr (9.5, black) compared to control (3, gray). (C) The individual effects in (A) and (B) combine to generate a colony-level quadrupling in the number of dances experienced in caffeine (black) versus control (gray) colonies for equal-molarity forage. Error bars represent SEM.
weight (kg honey stores). The results support the prediction such that with 40% caffeine patches in the environment, approximately 14.5% less honey will be produced each day compared to production by colonies foraging in a caffeine-free environment (Figure 4). Although evolutionary innovation often arises from genetic variation, new adaptations may additionally occur as a byproduct of unrelated processes, e.g., cooption or exaptation [19]. Caffeine occurs naturally in the leaves and seeds of many genera, including Citrus, Coffea, and Camellia, and possesses
a bitter taste that reduces palatability/digestibility to herbivores, thus acting as a chemical defense [3, 6, 7, 20]. The making and mobilization of these secondary compounds may have resulted in their accidental co-occurrence in nectar, where they may have subsequently been coopted for an additional role in plant reproduction [4, 5, 8, 21]. Nicotine, another secondary compound, has been shown to increase plant reproductive fitness by slightly decreasing nectar palatability to pollinators, who then move more quickly between flowers [22]. In this way, the effect of nicotine in nectar on pollinators mirrors its intended role in chemical
Figure 3. Caffeine Increases Persistency and Site Specificity (A) Initial persistency (number of visits per day to the now unrewarding feeder) significantly increased with caffeine, where bees receiving caffeine during training on day 0 (black line) visited 23% more compared to control (gray line) on day 1 of the feeder becoming unrewarding. Visits per day declined equally over time in both treatments. Dot size corresponds to sample size. (B) Caffeine significantly increases site specificity, such that a greater proportion of bees exposed to caffeine (58%, black) persistently returned to investigate only the unrewarding feeder to which they were initially trained compared to control bees (26%, gray), who were more likely to return and investigate both unrewarding feeders. Error bars represent SEM.
Current Biology 25, 1–4, November 2, 2015 ª2015 Elsevier Ltd All rights reserved 3
Please cite this article in press as: Couvillon et al., Caffeinated Forage Tricks Honeybees into Increasing Foraging and Recruitment Behaviors, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.08.052
Received: July 28, 2015 Revised: August 19, 2015 Accepted: August 24, 2015 Published: October 15, 2015 REFERENCES 1. Irwin, R.E., Bronstein, J.L., Manson, J.S., and Richardson, L. (2010). Nectar robbing: ecological and evolutionary perspectives. Annu. Rev. Ecol. Evol. Syst. 41, 271–292. 2. Dafni, A. (1984). Mimicry and deception in pollination. Annu. Rev. Ecol. Syst. 15, 259–278. 3. Berenbaum, M., and Rosenthal, G.A. (1992). Herbivores: Their Interactions with Secondary Plant Metabolites (Academic Press). 4. Wright, G.A., Baker, D.D., Palmer, M.J., Stabler, D., Mustard, J.A., Power, E.F., Borland, A.M., and Stevenson, P.C. (2013). Caffeine in floral nectar enhances a pollinator’s memory of reward. Science 339, 1202–1204. 5. Singaravelan, N., Nee’man, G., Inbar, M., and Izhaki, I. (2005). Feeding responses of free-flying honeybees to secondary compounds mimicking floral nectars. J. Chem. Ecol. 31, 2791–2804. 6. Kretschmar, J.A., and Baumann, T.W. (1999). Caffeine in Citrus flowers. Phytochemistry 52, 19–23.
Figure 4. Caffeine May Cause a Cost to the Colony Results of an agent-based model indicate that plants with nectar containing caffeine may lower their nectar sugar concentrations without decreasing their attractiveness to pollinators, resulting in colony weight loss. Caffeine, accompanied by a drop in nectar quality (black circle), is predicted to cause colonies to produce approximately 14.5% less honey per day compared both to colonies foraging in an environment with caffeine but without a drop in nectar quality (dark gray circle) and colonies foraging in an environment with no caffeine and no drop in nectar quality (light gray circle). Error bars represent SEM.
defense (e.g., decreasing palatability). In contrast, here we have shown that the effects of caffeine in nectar are akin to drugging, where the pollinator’s perception of the forage quality is altered, which in turn changes its individual behaviors. These behaviors may scale to colony-level effects, as predicted by the model, a hypothesis that could be experimentally tested. As such, these data provide a fascinating example of the ongoing tension between conflict and cooperation in mutualisms, and the diversity and subtlety of the mechanisms that are used to manipulate partners. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.cub. 2015.08.052. AUTHOR CONTRIBUTIONS M.J.C. and R.S. conceived and designed the experiment and oversaw data collection. M.J.C. wrote the manuscript. R.S. performed the statistical analyses. F.L.W.R. contributed valuable discussions and contributed to the writing of the manuscript. H.A.T., T.M.B., and F.S. assisted with data collection. ACKNOWLEDGMENTS M.J.C. is funded by the Nineveh Charitable Trust. T.M.B. is funded by the Natural Environment Research Council (NERC) and the University of Sussex. H.A.T. is funded by Damascus University. R.S. is funded by Rowse Honey Ltd. Additional research funding was provided by Waitrose Ltd. and Burt’s Bees.
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