Spontaneous quantity discrimination of artificial flowers by foraging honeybees

Many animals need to process numerical information in order to survive. Spontaneous quantity discrimination is useful for assessing food resources, aggressive interactions, predator avoidance, and prey choice. Spontaneous quantity discrimination is a numerical ability allowing differentiation between two or more quantities without reinforcement nor prior training on any numerical task. Honeybees have previously demonstrated the ability to learn to count landmarks, match quantities, use numerical rules, discriminate between quantities, and perform arithmetic, but have not been tested for spontaneous quantity discrimination. In bees, spontaneous quantity discrimination could be useful when assessing the quantity of flowers available in a patch and thus maximizing foraging efficiency. In the current study, we assessed the spontaneous quantity discrimination behaviour of honeybees. Bees were trained to associate a single yellow artificial flower with sucrose. Bees were then tested for their ability to discriminate between 13 different quantity comparisons of artificial flowers (numeric ratio range: 0.08 to 0.8). Bees significantly preferred the higher quantity only in comparisons where '1' was the lower quantity and with a sufficient distance between quantities (e.g. 1 vs 12 and 1 vs 4 but not 1 vs. 3). Our results suggest a possible evolutionary benefit to choosing a foraging patch with a higher quantity of flowers available only when resources are scarce.Funding provided by: Australian Government Research Training Program (RTP) Scholarship *Crossref Funder Registry ID: Award Number: Funding provided by: Fondation FyssenCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100003135Award Number: Funding provided by: Centre National de la Recherche ScientifiqueCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004794Award Number: Funding provided by: University of Toulouse 3Crossref Funder Registry ID: http://dx.doi.org/10.13039/501100004718Award Number: Funding provided by: Australian Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000923Award Number: DP130100015Funding provided by: Australian Government Research Training Program (RTP) ScholarshipCrossref Funder Registry ID:Study species Experiments took place at the Johannes Gutenberg University Mainz, Germany and the University of Melbourne, Australia in 2018 – 2019. Honeybee foragers (Apis mellifera (Linnaeus 1758)) were collected from either gravity feeders providing approximately 10 – 30 % by volume sucrose solution or directly from the hive entrances. Bees came from the managed hives of both universities. We tested 520 forager bees for their spontaneous preferences to larger or smaller quantities of artificial flowers and in control experiments. Apparatus Honeybees were trained to visit a rotating screen which presented four hangers at a time (Figure 1). Bees were individually recruited, trained, and tested. We used a standard rotating screen (Avarguès-Weber et al. 2010b; Dyer et al. 2005) which was 50 cm in diameter and was made of a grey plexi-glass material containing hanger pegs (Figure 1) which allowed hangers presenting stimuli to be attached. The hangers had landing platforms directly under stimuli where foragers could land to collect a drop of sucrose solution. The screen could be freely rotated between choices to randomise hanger positions. Additionally, hangers could be moved between pegs to change their positions. Once a bee had made a choice and landed, it was allowed to finish drinking the sucrose on the landing platform before being collected onto a plexi-glass spoon with sucrose solution and placed behind an opaque screen while landing platforms, hangers, and surrounding areas were cleaned with a 30 % ethanol solution, then water, and dried. New stimuli were then presented on the hangers and the screen was rotated to randomise the position of stimuli before the bee made another choice or returned to the hive if satiated. Priming phase Honeybee foragers were first trained to associate the artificial flower (yellow circle; Figure 1A) with a reward of sucrose. Bees were trained to land on the hangers of the rotating screen to receive a reward of sucrose. After bees learnt to individually land on the hangers, we placed an approximately 10 μL drop of 25 % sucrose solution on the hangers with one artificial flower directly above the landing platform containing the drop. Priming consisted of allowing bees to land and collect sucrose from hangers, each presenting a single yellow circle of differing size (six possible sizes from 1 cm2 to 10 cm2) (Figure 1A). The size of the priming stimuli could be pseudo-randomly changed when the bee returned to the hive, as a bee will return periodically to the hive once satiated (approximately after 2 – 6 choices known as a 'bout'). Once bees had made 20 landings to drink sucrose, thus associating the yellow circles with sucrose solution, they were given tests to determine if they would prefer to visit more yellow circles when given the option between two quantities of circles. Testing phase: Quantity comparisons Bees were tested for their preferences in 13 different quantity comparisons (Table 1) in the absence of reinforcement (sucrose). One group was tested using stimuli which had an overall equal surface area of yellow colour (10 cm2) regardless of element quantity and one group was tested using stimuli in which all individual elements were of equal size (all elements were 1 cm2). Low level cues such as surface area, convex hull, line length, and density are present when bees are foraging at flower patches. These non-numerical cues are correlated with increasing quantity and would be available to naturally foraging bees, thus we tested two conditions (surface area controlled or individual element size controlled) to begin to understand which cues bees may use for quantity tasks. Honeybees prefer to visit larger flowers (Martin 2004) and have preferences for specific shapes of flowers (Howard et al. 2018b; Lehrer et al. 1995), which may impact their choices of stimuli. Thus, the spatial arrangement of elements was randomised to account for this and different procedures were implemented to determine if they preferred larger stimuli (e.g. preference for lower quantities with larger stimuli in the equal surface area condition). This was done by testing each comparison with stimuli where there was an equal overall surface area of elements or where stimuli contained elements of the same size. If bees preferred larger artificial flowers, they should choose the lower number in the equal surface area condition, as these stimuli would have fewer elements with larger areas. If bees preferred a larger area of yellow, then they should consistently choose the larger number when elements were the same size. Each bee performed only one test comparison. The comparison the bee would be tested on was pseudo-randomly assigned and each test lasted for 10 choices each. Table 1: The quantity comparisons tested with the ratio, and the numeric value of the ratio shown for comparison purposes. For each comparison we report whether the quantities are within the OFS or AMS range or both. Also reported here is the number of bees tested per group, where 'Equal surface area is equal overall surface area of yellow area and 'Equal element size' is where all individual elements were of the same area. Quantities Numeric ratio Ratio OFS or AMS Number of bees tested Equal surface area Equal element size 1 vs. 12 0.08 1:12 Both 15 25 4 vs. 20 0.20 1:5 AMS 18 27 1 vs. 4 0.25 1:4 OFS 10 15 1 vs. 3 0.33 1:3 OFS 12 12 4 vs. 12 0.33 1:3 AMS 11 12 1 vs. 2 0.50 1:2 OFS 12 12 2 vs. 4 0.50 1:2 OFS 30 15 4 vs. 8 0.50 1:2 AMS 13 10 4 vs. 7 0.57 1:1.75 AMS 12 11 2 vs. 3 0.67 1:1.5 OFS 12 12 4 vs. 6 0.67 1:1.5 AMS 22 17 3 vs. 4 0.75 1:1.33 OFS 30 15 4 vs. 5 0.80 1:1.25 AMS 12 12 Testing phase: Control experiments Five different control experiments were conducted to ensure bees associated the yellow circle with sucrose solution and considered it as a rewarding 'flower'. These control experiments contained a priming phase of 20 landings and a testing phase of 10 choices. The first control experiment (control 1; n = 50) primed bees on the yellow circles, as above, and then tested bees on the comparison between a 6 x 6 cm yellow square vs. a 6 x 6 cm grey square, to determine if they had a preference for the colours used in the experiment after the same priming to artificial flowers. The second control experiment (control 2; n = 10), primed bees on the yellow circles, as above, and then tested bees on the comparison between a 6 x 6 cm grey card and 6 x 6 grey card containing a single yellow circle. This would determine if bees preferred the yellow circle over the grey card and thus associated the yellow circle with the reward. The other three control experiments (control 3 – 5), primed bees to sand-blasted grey aluminium 6 x 6 cm stimuli, then tested the bees on either 1 vs 12 (equal surface area stimuli; control 3; n = 34), 4 vs. 1 (equal surface area; control 4; n = 10), or 4 vs. 1 (equal element size; control 5; n = 12), to determine whether the priming to the single artificial flower was driving any preferences observed for quantities of artificial flowers. These control experiment comparisons were chosen as these tests in the quantity comparison phase of the experiments yielded significant results (see Table 2). Statistical analysis Ten choices per bee were recorded during the test and then analysed. To determine whether bees demonstrated a significant preference for a stimulus during the non-rewarded tests, we tested if the mean number of 'correct choices' observed for each comparison was significantly different from chance. We analysed data with a generalized linear mixed-effects model (GLMM) with a binomial distribution using the 'glmer' package within the R environment for statistical analysis (Team 2017). This was done by fitting individual GLM models to response data for each treatment including only the intercept term as a predictor