Bee Learning Lab

Abstract Bees use floral features to associate specific flowers with nectar rewards. With that being said, there is an array of floral features, like size and smell. Additionally, flower color is an easily identifiable feature as it is shown on the petals, which can be spotted quickly. However, in order for flower color to be distinguished, bees need to have the presence of color vision. To test whether bees have color vision, we designed an experiment that tested bees’ ability to associate a color with a nectar reward, through using conditioned stimuli, colored paper with sucrose solution, that was then tested through a test trial. The results of the test trial indicate that bees are able to correctly choose the color they were tested to, indicating that they have associatively learned a color that is paired with a reward. The presence of color association indicates that bees have color vision which adds to the decision making process of bees when foraging for nectar as they can associate color of a flower with levels of nectar. Introduction Bees are pollinators meaning they carry pollen from the male part to the female part of the flower allowing for flower fertilization during the process of retrieving nectar. Bees that forage for nectar visit many flowers, which vary in appearance and in the nectar reward. With that being said, bees do not select flowers randomly, rather they have preferences for flowers which guide the area they forage about (Heinrich, 1976). The term “floral constancy” refers to foraging animals, including bees, specializing on specific species of flowers when there is a mixture of species available (Wilson & Stine, 1995). Through floral constancy bees increase efficiency and nectar rewards as they do not waste time experimenting with many different species of flowers (Wilson & Stine, 1995). The species of flower bees are consistent with are influenced by floral features like size, shape, odor, and patterns (Hammer & Menzel, 1995). Bees use these floral components in their evaluation process in picking a site where these preferences are most prevalent as they can associate it with a nectar reward (Heinrich, 1976). Additionally, bees have innate, a psychological necessity, or learned preferences through associative learning which influences their foraging behavior. Associative learning is when a stimulus and a response are paired over time, so in the case of bees, a learned floral feature will be associated with nectar reward (Pontes et al., 2020). In previous work, conducted by Van Hateren et al., bees were able to recognize patterns by orientation through associative learning (Hateren et al., 1990). In that experimental study, bees were trained to learn different orientation patterns through getting a sugar water reward, indicating that bees were able to associate an orientation pattern with a sugar reward. With that being said, one of the outstanding features of a flower is their color, as it is the most visually drawing aspect of the plant that can be quickly identified. Since bees are flying pollinators, could seeing the color of flowers influence foraging patterns as color could be associated with nectar? To investigate if flower color can influence foraging patterns, we need to ask the question: do bees have color vision? To answer this question we collected data from 63 bees about their color choice in a test trial after training them with sugar water. We hypothesized that if bees can learn color associatively then we expect them to see color. However, if the hypothesis that bees can learn color associatively is false, color vision in bees is inconclusive. If our hypothesis that bees can associatively learn color is true, then we predict bees are able to see color. Methods Our experiment involved 63 bees, with one bee in a single test tube, blue and yellow strips of paper, a timing device, and sucrose solution. One female, naive, forager bee was placed per tube with two end holes, to which a colored strip of paper was placed. There were two treatments involved, each involved a different colored conditioned stimuli, a stimulus that causes a conditioned response. One treatment is the blue strip being the conditioned stimuli (CS+) and the other is the yellow strip being the conditioned stimuli (CS+) and the color of the CS+ was picked for us in relatively equal proportions. The conditioned stimuli was dipped in a 50% sucrose solution while punishing color (CS-) is dipped in water. There were 6 training rounds to which in each round the conditioned stimuli was placed at an end hole first, while the bee drank from her proboscis, her tongue, for 3 seconds. The same procedure was repeated but with the punishment color (CS-) which concluded one training round. Moreover, the next training round began after 5 minutes for 6 training rounds. The last part of the experiment was a test in which both strips of paper were placed in the end holes simultaneously but the conditioned stimulus has water not sucrose solution. Additionally, any behavioral observations were noted in a data sheet. In order to determine if color of CS+ influenced the test result, a chi-squared test was used to see if the two variables are independent from each other. Results During the color association training, bees were presented with a color stimulus (CS+) that had a sugar reward to which they were supposed to pick the color they were tested to for the test trial that had no sucrose solution. The distribution of bees that correctly selected the color they were tested to in the test trial is shown in Figure 1. Moreover, according to Figure 1, the majority of bees were able to develop a color association. More specifically, out of the 63 bees, 55 selected correctly, while 8 selected incorrectly. Additionally, Figure 2 indicates the frequency of incorrect and correct results by the CS+ color, which shows that there is a relatively equal number of bees between each CS+ for incorrect and correct results. Figure 2, further supports the hypothesis that bees can see color, since there was a statistically significant positive relationship between incorrect and correct responses by CS+ color, X2 (df=1, N=63) = 35.06, p < .05. Therefore, it can be said that the statistically significant positive relationship indicates that bees did not pick the correct result during the test trial due to random chance. Rather, the bees had a learned association, for both CS+ colors as seen in Figure 2, supporting the hypothesis that bees can see color, as bees cannot associate color with a reward without the presence of color vision. Discussion There have been studies demonstrating the ability of bees to recognize many floral features. Recently, there has been a study illustrating that bumblebees have a higher preference for flowers with a higher floral humidity, higher humidity around the headspace of a flower (Harrap et al., 2021). This study indicates that bees have a high sense of awareness when foraging and many attributes of a flower influence flower visitations. Another study proves that there is an innate behavior for bees to prefer bilateral symmetry for flowers, indicating that bumblebees do have the ability to recognize symmetry (West & Laverty, 1998). Thus, if bees are able to recognize such specific and unique parts of a flower, the question arises if they are able to see color, a visually obvious portion of a flower. Like in the other experiments, similar methodologies were used, training trials with conditioned stimuli that had sucrose water and a test trial. With that being said, in our experiment it was demonstrated that bees do have the ability to see color, which may influence their foraging behaviors as they can associate flowers to nectar rewards. Thus, this experiment further proves bees’ ability to discriminate floral features of a flower, specifically color. It is important to note that our experiment did only test two colors in the conditioned stimuli, blue and yellow. Furthermore, bees have photoreceptors in UV, green, and blue while humans have photoreceptors in blue, green and red (Hempel et al, 2014). Because bees have different photoreceptors there is a possibility that they are not able to see some colors of the spectrum or different hues and brightnesses that humans may see. Thus, this study is not representative of the extent of color vision in bees, but only the presence of some degree. If we were to conduct this experiment again or further investigate, there should be more conditioned stimuli of colors that range in hues and brightnesses. Bees are flying pollinators that forage for nectar so it is important for them to identify different floral components so they can associate nectar reward by floral features. With that being said, the color of a flower is an obvious feature of a flower so color vision in bees can be associated quickly, especially while flying. Color vision adds another floral feature that bees can use to evaluate which flowers would present the best nectar reward. Additionally, now that it is known that bees can see color, we suggest that further investigation should be conducted to determine to what extent bees can see color, if tone, hue, and brightness affect vision. References Harrap, M. J., de Ibarra, N. H., Knowles, H. D., Whitney, H. M., & Rands, S. A. (2021). Bumblebees can detect floral humidity. Journal of Experimental Biology. https://doi.org/10.1101/2021.03.19.436119 Hateren, J. H. V., Srinivasan, M. V., & Wait, P. B. (1990). Pattern recognition in bees: orientation discrimination. Journal of comparative physiology a-Sensory neural and behavioral physiology, 167(5), 649-654. Heinrich B (1976) The foraging specializations of individual bum- blebees. Ecol Monogr46:105- 128 Hempel de Ibarra N, Vorobyev M, Menzel R. Mechanisms, functions and ecology of colour vision in the honeybee. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2014 Jun;200(6):411-33. doi: 10.1007/s00359-014-0915-1. Epub 2014 May 15. 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