Experimental Setup and Initial Dataset
The transcriptomic data used for analysis was part of an experiment prepared to study the molecular basis for social learning of distance in honeybees through the waggle dance . In this experiment honeybees from 3 different colonies were trained to visit a feeder positioned at the end of a 6m long tunnel , which was used to alter the bee’s perception of distance as follows: vertical stripes (with respect to the direction of flight) on the tunnel walls were used to increase the estimated flight distance, while horizontal stripes were used to decrease it (Figure 1). Honeybees were then marked at the feeder according to perceived distance (similarly to , yielding two groups: “honeybees perceiving long distance” and “honeybees perceiving short distance”.
Honeybee colonies were housed in an observation hive, which allowed direct monitoring of the comb where honeybees normally performed the waggle dance after returning from a foraging trip, known as the “dance floor” . Bees were trained during the morning to visit the feeder at the end of the tunnel – it usually took approximately 5 hours to complete this part – and then in the afternoon (between 2pm and 4pm) foragers that regularly visited the feeder were monitored by an observer while flying from the dance floor to the feeder and vice-versa repeatedly. During this 2-hour time window, the dance floor was also recorded with a video camera, producing a recording of all waggle dance events that occurred in the focal colony. The footage was then carefully analysed to identify marked honeybees that performed waggle dances upon their return from the feeder (from now on “dancers”) and separate them from those that instead were never seen performing any dance (“non-dancers”) despite being visible on the dancefloor upon their return from a foraging trip. An analysis of the recorded dances confirmed that the manipulation was successful: bees exposed to vertical stripes advertised longer distances on average in their dances compared to bees exposed to horizontal stripes (Manfredini et al. in prep ).
This resulted in the following 4 groups of honeybees: Dancer perceiving Long distance (DL), Dancer perceiving Short distance (DS), Non-dancer perceiving Long distance (NL), Non-Dancer perceiving Short distance (NS), with 8 replicate samples in each of the 4 groups (N = 32). Brain tissues from all these samples were processed individually for RNAseq analysis (see Supplemental Information). RNAseq read files were aligned to the most recent version of the A. mellifera genome (Amel_4.5) using the intron-aware STAR aligner version 2.6.1a . Read counts were extracted using the featureCounts function from the Bioconductor R package ‘Subread’ version 1.8.0 . The final dataset, which represent the starting material for this study, included the read counts for 15,314 genes, corresponding to the whole honeybee genome across 32 bees. As we noticed some variation in library size for some of the bee samples, we normalised read counts by the total library sizes to correct for the effect of possible outliers.