A small brain and short life allegedly limit cognitive abilities. Our view of invertebrate cognition may also be biased by the choice of experimental stimuli. Here, the stimuli (color) pairs in Match-To-Sample (MTS) tasks affected the performance of buff-tailed bumblebees (Bombus terrestris). We trained the bees to roll a tool, ball, to a goal that matched its color. Color-matching performance was slower with yellow-and-orange/red than with blue-and-yellow stimuli. When assessing the bees' concept learning in a transfer test with a novel color, the bees trained with blue-and-yellow (novel color: orange/red) were highly successful, the bees trained with blue-and-orange/red (novel color: yellow) did not differ from random, and those trained with yellow-and-orange/red (novel color: blue) failed the test. These results highlight that stimulus salience can affect the conclusions on test subjects' cognitive ability. Therefore, we encourage paying attention to stimulus salience (among other factors) when assessing invertebrate cognition.

Study system

The experiments were conducted in 2018 in bumblebee facilities at the Botanical Garden of the University of Oulu, Finland. We obtained bumblebees from a continuous rearing program (Koppert B.V., The Netherlands). Each of the bumblebee hives (N = 7) used in the study were housed in a wooden box (31 cm × 13.5 cm × 11.5 cm) that had holes for air exchange and separate entrance and main hive chambers, with a 3 cm layer of cat litter at the bottom of the former. Each hive had a queen and ~30 workers. We provided each hive with ~7 g commercial pollen (Koppert B.V., The Netherlands) on every second day and, when not being trained or tested (see below) in which the bees had a continuous opportunity to forage on a 30% sucrose solution from a feeder.

We used one hive at a time. Its entrance chamber was connected to a transparent plexiglass corridor (25 cm × 4 cm × 4.5 cm), which allowed bumblebees to access an arena (60 cm × 25 cm × 43 cm). Three transparent plastic sliding doors along the corridor provided means to control the access of bumblebees to the arena (for testing purposes). This setup was used during pretraining, training and testing (see below).

Pretraining

The aim of this pretraining was to allow the bees to learn the location where to access the reward. In the pretraining, the bumblebees had unrestricted access to the arena where they could access 30% sucrose solution from the middle of a circular white platform (Ø 150 mm) that was placed in the central part of the arena. During the pretraining, the most active foragers were identified by an observer (OJL and VN) and each of these bees was marked with a small number tag. These tagged individuals were used in the training and test.

Training

The purpose of the training was to assess our hypothesis, while training the bees to match to a sample. In the training, the center of the arena had a white circular plastic platform (Ø 150 mm with a bordering wall 12 mm high). This platform had a hole in its center (Ø 12 mm) and a colored circular zone encompassing the center hole (Ø 35 mm). Three lanes (20 mm wide at the center section, outlined by 1 mm high and 10 mm wide plastic strips) ran from the rim of the platform and converged at the central zone at 120° angles relative to the adjacent lanes (Figure 1A). The platform also had two wooden balls (Ø 8.5 mm) of different colors (blue and yellow, blue and orange/red, or yellow and orange/red), painted using Uni POSCA PC-5M, Mitsubishi Pencil Co., LTD. Japan (Figure 1). The bordering wall, the three lanes, the circular zone around the center hole (collectively referred to as 'platform'), and one of the two balls had the color that matched the platform, while the other ball was of a different color.

During the training, only one tagged bee (N = 28 over the experiment) was allowed to access the arena at a time. Each bee was randomly assigned to a treatment group (blue and yellow, blue and orange/red, or yellow and orange/red) and thus only exposed to only two of the three colors used in the experiment. In each treatment group, a bee was exposed to the platform of two different colors. There were two balls, with one of the two balls always matching the color of the platform and the other ball having the other color. Each bee was challenged with a color matching task in the context of token use. The bee was given 5 minutes to complete a training bout. During a training bout, the 'correct' (rewarding) action required the bumblebee to successfully roll the ball that matched the color of the platform, from the rim of the platform to its center hole. Rolling the ball onto the central zone surrounding the hole, but not all the way into the hole, was also considered as successful. If the bee was successful, the experimenter used a syringe to immediately place a reward of 30% sucrose solution ad libitum (>200 µl) in the central hole for the bee to drink. Failing to accomplish the task within 5 minutes (or rolling the non-matching ball onto the central zone) was deemed as ‘incorrect’ (i.e., the bumblebee did not accomplish the task). After each training bout (successful or fail), the bumblebee was allowed to use the connecting corridor to visit the hive and then later to return to the arena to try again (i.e., the start of another training bout). We cleaned both balls and the platform cleaned with ethanol to neutralize any odor cues after each training bout. We also switched the color of the platform's color theme between the two options used for that particular bumblebee after every 1-3 training bouts. The behavior of the bee was video recorded for later analysis using a Sony Xperia XZ Premium smartphone. As soon as an individual reached the criterion of training (matching the ball that had the color as the platform for 5 or more consecutive bouts), she was used in the transfer test. At the end of each day, all the bees were allowed to freely access the arena to forage from a white platform, as during the pre-training phase.

The training progressed in a stepwise fashion that included four steps. In the first step, the ball that matched the color of the platform was already in the central hole, and the bumblebee was rewarded as soon as it touched that ball. Once this had happened, the task progressed to the second step, in which the 'correct' ball was placed next to the central zone. After this step was successfully completed, the third step involved the balls being placed in the midway between the central zone and the rim of the platform. Once the focal bumblebee completed this step, the final step involved both balls being placed at the rim of the platform, from where the bumblebee needed to roll the ball to the center. Most, if not all, of the bees failed one or more steps during the training. When a bumblebee did not successfully perform the task correctly within 5 minutes training bouts, the experimenter (OJL and VN) used a plastic model bumblebee (which mimicked the color patterns of a B. terrestris worker), attached to a thin transparent stick, to demonstrate how to solve the task. The experimenter then used a syringe to give the sucrose solution directly to the bumblebee. A model, rather than living, bumblebee demonstrator ensured a desired and standardized demonstration.

Transfer test

The purpose of the transfer test was to assess whether the bees exhibit concept learning by applying a learned rule in a novel context. The test was conducted once a bee reached the training criterion (5 or more successful training bouts in a row). The test consisted of a single bout that was similar to the last phase of training with the following exceptions: The platform was of the 'third' color that the bumblebee had not encountered during the training. In addition, the platform had 3 balls of different colors: blue, yellow or orange/red. One ball was placed at the end of each lane, next to the rim of the platform. The test ended if the bee rolled the correct ball to the central hole. If the bumblebee rolled a ball of a color that did not match with that of the platform, it was considered to have failed the test and the test continued during which the 'incorrect' ball was returned to the trim of the platform until 10 minutes passed.

QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analyses were conducted using R version 3.6.2 and SPSS v25 (IBM Corp). Generalised Linear Mixed Models (GLMM) with a poisson distribution (link = log) in the package 'glmmTMB' were used to examine whether the color pair (three levels: blue-and-yellow, blue-and-orange/red, and yellow-and-orange/red) affected the number of bout taken to reach the training criterion. We included bee ID nested within colony ID as the random variable.

To assessing whether bumblebees learned to solve the generalization task, we used 1/3 as the baseline expectation and compared it to bumblebees' performance (in terms of the number of bumblebees that solved vs. did not solve the task) for each of the treatment group using a binomial test. To examine the effect of the color pairs in relation to success in the test, we conducted a GLMM with binomial distribution (link = logit). However, due to convergent issues (likely related to the zeros, or the bees in the yellow-and-orange/red treatment group completely failed the test), the model could not be run. Accordingly, we compared bumblebees’ test performance between the three treatment groups using Fisher’s exact test. We also used Fisher’s exact test with Bonferroni corrections for posthoc analyses, when comparing the performance between any two treatment groups (adjusted significance level: P≤0.017).

Another GLMM with poisson distribution (link = log) was conducted to examine whether the training bouts differed between the bees that have successfully completed the transfer test and those bees that failed the test. For this analysis, we included colony, bee ID and the color pairs as the random variables. Given that the treatment groups differed in their training bouts to reach the criterion (Figure 2C-D), we further conducted three GLMMs with poisson distribution (link = log) to examine whether the bees that had successfully completed the transfer test differed from those that had failed the test between treatment groups using separate analyses. In these models, we included colony and bee ID as the random variables. We adjusted the P values using Bonferroni correction (P = 0.017) for multiple comparisons.

To further examine the bees that have failed in the blue-and-yellow group, we conducted beta regression using the package 'betareg'. One analysis was conducted to examine whether the bees that failed the test spent a higher proportion of time interacting with the incorrect balls than the correct ball. Another analysis was conducted to compare whether the bees that failed the test spent a higher proportion of time interacting with the incorrect balls than the bees who succeeded the test. Beta regressions were also used to analyze the proportion of time spent on interacting with the balls for the two yellow-and-orange/red groups. We compared the proportion of time spent on interacting with the incorrect balls and the correct ball for the 1st yellow-and-orange group in one analysis, and another analysis for the 2nd yellow-and-orange group.

Other than the posthoc tests using Bonferroni corrections with adjusted P values, we consider each test reached a significance when α<0.05 (two tailed). All results reported here used bootstrapped methods (10000 random replicates) on raw data to obtain means and 95% confidence intervals.

To visualize the colors of the platforms and balls as perceived by bees (Figure 1B), we used the color hexagon model with the vismodel function using the package 'pavo' in R.