Article Figures & Data
Figures
- Figure 1.
Calcium signals from m-ALT and l-ALT PNs to pheromonal odorants. A, Dual olfactory pathway of the honey bee brain (adapted from Carcaud et al., 2012). Odorant molecules are detected by ORNs on the antenna, forming the antennal nerve (AN) and project to the AL. Then, PNs convey information to the MB and the LH via two main tracts: the m-ALT (magenta) and the l-ALT (green). Injection locations are shown as black dots (inj1 and inj2). OL, Optical lobe. B, Retrograde staining of l-ALT PNs (left) and m-ALT PNs (right) innervating, respectively, ventral and dorsal AL glomeruli. Z-projection of optical slices at the indicated depths. C, Odor-induced calcium signals in the AL at room temperature (∼25°C, left) or at hive temperature (35°C, right), for l-ALT PNs to QMP (top) and for m-ALT PNs to methyl linolenate (bottom). Relative fluorescence changes (ΔR/R%) are presented in a false-color code, from dark blue to red. D, Time course of ΔR/R% during a 20 s 5 Hz recording for l-ALT (top) and m-ALT PNs (bottom). The signals from different glomeruli (1–4) located as indicated in C are shown for room T° (dashed lines) and hive T° (solid lines).
- Figure 2.
Responses to queen and brood pheromone compounds in l-ALT and m-ALT PNs. A, Odor-induced calcium activity maps obtained from l-ALT (top) and m-ALT neurons (bottom) in two different animals, to 5 queen mandibular compounds and the QMP mixture, to 3 queen nonmandibular compounds, to 6 compounds of the brood pheromone and to the solvent. Relative fluorescence changes (ΔR/R%) are presented in a false-color code, from dark blue to red. B, Pixelwise analysis. Amplitude of calcium responses (ΔR/R%) in the antennal lobe for m-ALT (n = 9) and l-ALT neurons (n = 10) to queen mandibular compounds (in red), queen nonmandibular compounds (in orange), and brood pheromone compounds (in green). Gray represents the response to the solvent. C, Glomerular analysis. Example of repartition of the 20 glomerulus-sized areas of interest (henceforth “glomeruli”) on a l-ALT recording, showing overlay with 3 odor response maps. D, Number of activated glomeruli in m-ALT (n = 9) and l-ALT neurons (n = 10). Error bars indicate the SEM across animals. Stars on the right of each bar indicate significant difference compared with solvent (Dunnett's test). *p < 0.05; **p < 0.01; ***p < 0.001; (*)p < 0.1.
- Figure 3.
Coding of queen and brood pheromone compounds in both subsystems. These analyses are based on pixelwise Euclidian distances between odor representations after averaging across animals, for l-ALT (left) and m-ALT neurons. A, Cluster analysis showing similarity relationships among representations of queen and brood pheromone compounds (Ward's method). Higher linkage distances correspond to more dissimilar odorants. Red represents queen mandibular compounds; orange represents queen nonmandibular compounds; green represents brood pheromone compounds. Gray represents the solvent. Gray background represents noncoding compounds within each neuron type (Fig. 2B). White background represents compounds processed within each subsystem. B, Proximity analysis (multidimensional scaling). The two main dimensions explain 69% and 76% of overall variance for l-ALT (left) and m-ALT neurons (right), respectively.
- Figure 4.
Representation sparseness and glomerular coactivation for queen and brood pheromone. A, Population sparseness and lifetime sparseness measured from l-ALT and m-ALT recordings to queen and brood pheromones. Boxes represent the median and first and third quartiles. Whiskers represent 10% and 90% percentiles. ***p < 0.001. B, Glomerular coactivation. Principle for the classification of glomeruli according to four classes depending on their coactivating odorants from the same and/or another pheromone (putative example). For each reference odorant, all activated glomeruli were classified as follows: unique glomerulus (white arrow, only activated by the reference odorant), same type glomerulus (light gray arrow, activated by the reference odorant and by another odorant of the same pheromone), other type glomerulus (dark gray arrow, activated by the reference odorant, not by other odorants of the same pheromone but activated by other pheromone types; here Pheromones B and C), or mixed glomerulus (black arrow, activated by odorants from both the same and other pheromone types). C, Proportions of the four classes of glomeruli for each pheromone compound and for all the glomeruli they activate: white represents unique; light gray represents same type; dark gray represents other type; black represents mixed.
- Figure 5.
Responses to alarm and aggregation compounds in l-ALT and m-ALT PNs. A, Odor-induced calcium activity maps obtained from l-ALT (top) and m-ALT neurons (bottom) in different animals to 9 alarm compounds (in blue), 4 aggregation compounds (in brown), and to the control (air). Relative fluorescence changes (ΔR/R%) are presented in a false-color code, from dark blue to red. B, Amplitude of calcium responses (ΔR/R%). C, Numbers of activated glomeruli recorded for l-ALT (n = 10) and in m-ALT neurons (n = 8) to alarm (in blue) and aggregation compounds (in brown). Gray represents responses to the air control. Error bars indicate SEM. D, Correlation between the amplitudes of calcium responses (ΔR/R%) recorded in the l-ALT and m-ALT neurons for alarm and aggregation compounds (black circles and solid line) and for queen mandibular, nonmandibular, and brood compounds (gray circles and dashed line). Dunnett's test: *p < 0.05; **p < 0.01; ***p < 0.001; (*)p < 0.1.
- Figure 6.
Coding of alarm and aggregation pheromone compounds in both subsystems. A, Cluster analysis showing similarity relationships among alarm and aggregation compounds (Ward's method), using Euclidian distances obtained for the 91 odor pairs in both subsystems. In both analyses, blue represents alarm compounds, and brown represents aggregation compounds. Gray represents air control. Gray background represents noncoding compounds within each neuron type (Fig. 5B). White background represents the compounds processed within each subsystem. The dendrogram for l-ALT neurons (left) does not show a separation based on the pheromonal nature of the compounds. Rather, short chain alcohols are separated from most other compounds. The cluster analysis for the m-ALT neurons (right) isolates aggregation compounds with the alcohols of the alarm pheromone from the remaining alarm compounds. B, Proximity analysis for both neuron types. For l-ALT neurons (left), the first dimension (46% of variance) separates processed from the nonprocessed compounds. The second dimension (24% of variance) separates acetate esters from short chain alcohols. Proximity analysis for m-ALT neurons (right) defines two main dimensions (75% of variance), which mostly differentiated ester-type from alcohol-type alarm compounds, the latter appearing together with aggregation compounds.
- Figure 7.
Representation sparseness and glomerular coactivation for alarm and aggregation pheromone. A, Population sparseness and lifetime sparseness measured from l-ALT and m-ALT recordings to alarm and aggregation pheromone compounds. Boxes represent the median and first and third quartiles. Whiskers represent 10% and 90% percentiles. **p < 0.01. B, Glomerular coactivation. Representation for each compound, of the percentage of glomeruli observed in the four classes (for definitions, see Fig. 4B): white represents unique; light gray represents same type; dark gray represents other type; black represents mixed. Only compounds that induced significant activity in l-ALT (top) or m-ALT neurons (bottom) are represented. The majority of glomeruli are mixed glomeruli, being activated both by other compounds of the same pheromone and by other odorants.
Tables
Pheromone type Compound Abbreviation Vapor pressure (mmHg) Queen mandibular compounds (QM) Homovanillyl alcohol HVA 2.04 × 10−5 Methyl 4-hydroxybenzoate HOB 2.37 × 10−4 9-Oxo-(E)-2-decenoic acid 9-ODA 1.77 × 10−6 9-Hydroxy-(E)-2-decenoic acid 9-HDA 2.87 × 10−6 10-Hydroxy-(E)-2-decenoic acid 10-HDA 6.28 × 10−6 Queen nonmandibular compounds (QnM) Coniferyl alcohol — 8.80 × 10−6 1-Hexadecanol 1–16ol 6.00 × 10−6 Linolenic acid — 5.40 × 10−7 Brood compounds (brood) Methyl linoleate — 3.67 × 10−6 Methyl linolenate m. linolenate 5.07 × 10−6 Ethyl oleate — 6.07 × 10−5 Methyl oleate — 6.29 × 10−6 Ethyl palmitate — 2.34 × 10−5 Methyl palmitate — 4.70 × 10−5 Alarm compounds (alarm) Isopentyl acetate IPA 5.60 2-Heptanone 2–7one 3.86 1-Butanol 1–4ol 6.70 1-Hexanol 1–6ol 0.93 1-Octanol 1–8ol 7.94 × 10−2 2-Nonanol 2–9ol 6.76 × 10−2 Octyl acetate — 0.22 Benzyl acetate — 0.17 Hexyl acetate — 1.32 Aggregation compounds (aggregation) Geranic acid — 3.27 × 10−3 Farnesol — 2.52 × 10−4 Citral — 9.13 × 10−2 Geraniol — 3.00 × 10−2
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