Does Fine Color Discrimination Learning in Free-Flying Honeybees Change Mushroom-Body Calyx Neuroarchitecture?

Abstract

Honeybees learn color information of rewarding flowers and recall these memories in future decisions. For fine color discrimination, bees require differential conditioning with a concurrent presentation of target and distractor stimuli to form a long-term memory. Here we investigated whether the long-term storage of color information shapes the neural network of microglomeruli in the mushroom body calyces and if this depends on the type of conditioning. Free-flying honeybees were individually trained to a pair of perceptually similar colors in either absolute conditioning towards one of the colors or in differential conditioning with both colors. Subsequently, bees of either conditioning groups were tested in non-rewarded discrimination tests with the two colors. Only bees trained with differential conditioning preferred the previously learned color, whereas bees of the absolute conditioning group, and a stimuli-naïve group, chose randomly among color stimuli. All bees were then kept individually for three days in the dark to allow for complete long-term memory formation. Whole-mount immunostaining was subsequently used to quantify variation of microglomeruli number and density in the mushroom-body lip and collar. We found no significant differences among groups in neuropil volumes and total microglomeruli numbers, but learning performance was negatively correlated with microglomeruli density in the absolute conditioning group. Based on these findings we aim to promote future research approaches combining behaviorally relevant color learning tests in honeybees under free-flight conditions with neuroimaging analysis; we also discuss possible limitations of this approach.

Fig 1. Color stimuli qualities.

A Spectral reflectance of stimuli. B Loci of color stimuli in a color hexagon. The cross marks the location of the blue stimulus, while the triangle indicates the location of the turquoise stimulus. The hexagon’s center is indicated by the dot. See text for details.

Fig 2. Synapsin immunostaining and 3D calyx reconstruction of a forager honeybee brain.

A Confocal image of a frontal section through the brain after whole-mount immunolabeling for synapsin. Calyx volume and MG density were quantified in one of the medial calyces (mCA). In the magnified view of the lip (B) and the collar (C), synapsin-labeled projection neuron boutons (MG) were counted in defined volumes (1000 μm³; yellow cubes) in three regions: mALT innervated lip, lALT innervated lip, and dense collar (dCO). D Cross section of the volume reconstruction of the mCA rendered from confocal image stacks. AL, antennal lobe; BR, basal ring; mCA, medial calyx; lCA, lateral calyx; CX, central complex; dCO, dense collar; lCO, loose collar; LA, lamina; LO, lobula; MB, mushroom body; ME, medulla; PED, peduncle. Axes: ca, caudal; le, left; ri, right; ro, rostral. Scale bar in A is 500 μm, in B (and C) 25 μm, and in D 100 μm.

Fig 3. Learning performance and color discrimination (choice) test of bees of the three experimental groups.

One group of bees was trained with absolute conditioning to one color (against the grey background, black squares). A second group received differential conditioning with one color rewarded and a second color unrewarded (open squares). A third (control) group experienced the training without conditioning to color stimuli (grey squares). See material and methods for the definition of “correct” decision in the grey control group. All groups completed 50 conditioning trials, followed by a choice test, where all bees had to choose between the two colors used in the experiment (blue and turquoise). Horizontal grey dashed line indicates chance level (random choice). All values are mean proportion (±SEM) of correct decisions; **p<0.01; ***p<0.001; ns: not significant; absolute: N = 15; differential: N = 16; grey: N = 12.

Fig 4. Volume of the entire median MB calyx and MB calyx subcompartments (dense collar and lip).

No differences were found among experimental groups for the volumes of the entire calyx, dense collar and lip regions. ns: not significant; absolute: N = 13; differential: N = 14; grey: N = 10; feeder: N = 10.

Fig 5. Number of microglomeruli per cube (10x10x10μm in size, A) and extrapolated number of MG per calyx (B).

No differences in MG numbers were found among groups in any region (dense collar, lip), either when counted per cube or extrapolated to the total number (per calyx). ns: not significant; absolute: N = 13; differential: N = 14; grey: N = 10; feeder: N = 10.

Fig 6. Correlation between numbers of microglomeruli (MG) and numbers of correct landings in absolute conditioning experiment.

Evidence for a correlation between the absolute number of correct landings (from a total of 50 decisions during color conditioning) and an individual’s MG number in both calyx subregions, the lip and the dense collar. Average number of MG per 1000 μm³ cube (A) and extrapolated to whole volume (B). N = 13.