Genetically encoded GCaMP6f and its neural expression.

(A) Scheme of the honey bee synapsin promoter GCaMP6f expression cassette. CDS: coding sequence; UTR: untranslated region; ATG: translation start. (B) GCaMP6f expression in the honey bee brain revealed by anti-GFP immunostaining (left, in green). GCaMP6f is widely expressed in the bee brain, including in somata and neural tracts (white arrows). For comparison with synapsin expression, an anti-SYNORF1 immunostaining (right, in red) is superimposed on the anti-GFP signal in green. Scale bar = 200 μm. (C) GCaMP6f expression (anti-GFP in green) and synapsin expression (anti-SYNORF1 in red) in the antennal lobe. Remarkable and strong expression is observed in the somata of projection neurons and local neurons (near the AL, white arrows). Scale bar = 50 μm. (D) GCaMP6f expression (anti-GFP in green) and synapsin expression (anti-SYNORF1 in red) in the mushroom bodies with strong expression in some somata of Kenyon cells (in the cup of the calyces, see white arrows). Scale bar = 50 μm. AL: antennal lobe, MB: mushroom body, OL: optic lobe, Lo: lobulla, Me: medulla, vL: vertical lobe, lc: lateral cluster of antennal lobe neuron somata, mc: medial cluster of antennal lobe neuron somata, Li: lip, BR: basal ring, Co: collar.</p

Neural activity recorded in the LH.

(A) Calcium signals in the LH evoked by the same panel of 16 odorants. Different odorants induce different activity patterns in the LH. (B) Example time courses (top, taken from the black square shown in A in C7 ketones) and average time courses (bottom, n = 8 honey bees) of odor-evoked responses (ΔF/F [%]) recorded in the LH (in the black square shown in A) to 2-heptanone (in red), octanal (in blue), and to the air control (in gray). The calcium signals also show a biphasic response, with a fluorescence increase upon odor presentation (blue bar) followed by a long undershoot. (C) Amplitude of calcium responses (ΔF/F [%]) to the 16 aliphatic odorants and to the air control. All odorants induce a significant activity in comparison to the air control (n = 8 honey bees, * p D) Cluster analysis showing similarity relationships among odorants (Ward’s classification method). The data underlying the graphs shown in the figure can be found in S2 and S6 Data files. LH, lateral horn.</p

Calcium imaging data recorded in the antennal lobe.

Data underlying Fig 3. (XLSX)</p

Calcium imaging data recorded in the lateral horn in response to pheromonal compounds.

Data underlying Fig 6. (XLSX)</p

Calcium imaging data recorded in the mushroom bodies.

Data underlying Fig 5. (XLSX)</p

Behavioral Euclidian distances.

From Guerrieri and colleagues [49]. Data underlying S6 Fig. (XLSX)</p

Correlation between amplitudes of calcium responses recorded in the antennal lobe.

(A) Amplitude of calcium responses (ΔF/F [%]) recorded in OSNs in [45] as a function of the amplitude of calcium responses (ΔF/F [%]) recorded in the AL in this study. The linear regression shows a significant correlation (R2 = 0.84, *** p = 6 × 10−7). (B) Amplitude of calcium responses (ΔF/F [%]) recorded in PNs in [19] as a function of the amplitude of calcium responses (ΔF/F [%]) recorded in the AL in this study. The linear regression shows a significant correlation (R2 = 0.78, *** p = 6 × 10−6). (TIF)</p

GCaMP6f expression in different regions.

(A) GCaMP6f expression (anti-GFP in green) in the lateral cluster of local/projection neurons near the AL (left) and its zoom (right), showing very broad expression in almost all the somata. Scale bar = 50 μm. AL: antennal lobe. (B) GCaMP6f expression (anti-GFP in green) in the photoreceptors of the ocelli. MB: mushroom body. (TIF)</p

Time courses of odor-evoked responses recorded in the lateral horn.

Time course of odor-evoked responses (ΔF/F [%]) recorded in the LH (n = 8 honey bees) to the different odorants and to the air control (in gray). The data underlying the graphs shown in the figure can be found in S6 Data. (TIF)</p

Correlation between neural and behavioral distances.

Correlation of Euclidian distances recorded in the AL in this study are highly correlated with behavioral distances recorded in (48) (R2 = 0.34, *** p S8 Data. (TIF)</p