Fig 1.
AmBRP variants detected in the honeybee brain.
a Map of the BRPlast200 and the BRPD2 antibodies’ epitopes in the large Drosophila BRP isoforms D (NP_724796). b Immunoblot of honeybee central brain and fruit fly head homogenate. The BRPlast200 antibody and the BRPD2 antibody recognize two bands around 220 kDa in honeybee central brains (lane 2, 4), and two major bands and several light bands between 120 and 220 kDa in fruit fly heads (lane 3, 5).
Table 1.
Primary antibodies used in this study.
Fig 2.
Distribution of AmBRP and Synapsin in the honeybee central brain.
Optical sections of a honeybee central brain incubated with the anti-BRPlast200 and anti-SYNORF1 to visualize the presynaptic proteins AmBRP and Synapsin. a Longitudinal section through a schematic honeybee brain showing ventrally located regions (nomenclature after Ito et al. (2014) [34]). b-d Distribution of BRPlast200 signals (b) and anti-SYNORF1 signals (c) in ventrally located brain regions of a 29-day-old bee. Both antibodies show staining in all brain regions with almost similar distribution (d). Prominent stained regions are the vertical lobes and the antennal lobes. e Longitudinal section through a schematic honeybee brain showing dorsally located regions (nomenclature after Ito et al. (2014) [34]).f-h Distribution of BRPlast200 signals (f) and anti-SYNORF1 signals (g) in dorsally located brain regions of a 29-day-old bee. Both antibodies show staining in all brain regions with similar distribution (h) Prominent stained regions are the peduncles, especially in the AmBRP staining. LCA, lateral calyx; MCA, medial calyx; VL, vertical lobe; PED, peduncle; ML, medial lobe; CB, central body; AL, antennal lobe; NA, neuraxis anterior; M, medial; NP, neuraxis posterior; L, lateral. Scale bars: 200 μm for b-d and f-h.
Fig 3.
AmBRP is predominantly located in the vicinity of the membrane of presynaptic boutons within microglomeruli in the mushroom body calyces.
a-c Confocal images of the medial calyx showing BRPlast200 staining (AmBRP, green, b) in combination with Phalloidin staining (F-actin, magenta, a) and an anti-SYNORF1 counterstaining (Synapsin, blue, a) to visualize pre- and postsynaptic structures in a 8-day-old bee. The calyx can be subdivided into three regions, lip, collar and basal ring. Experiments focused on the lip and the dense region of the collar (dCO). d Schematic representation of a microglomerulus (MG) (modified after [35] showing already established pre- and postsynaptic marker (Synapsin, blue; F-actin, magenta). The bouton of a projection neuron is surrounded by spines from Kenyon cell dendrites. Anti-SYNORF1 labels the vesicle-associated protein Synapsin (blue) whereas Alexa Fluor 546 Phalloidin binds to F-actin located in dendritic spines (magenta). e-k Confocal images of MG in the dense collar region with labeled Synapsin (e), F-actin (f) and AmBRP (h). The F-actin signals form circles around Synapsin signals (g). AmBRP is located predominantly at the outer rim of the Synapsin-labeled signals and at the inner rim of the F-actin signals (i-k). The insets show a single, magnified MG from the corresponding image. LI, lip; CO, collar; BR, basal ring; dCO, dense collar; PN, projection neuron; KC, Kenyon cell.Scale bars: 20 μm for a-c, 2 μm for e-k, 1 μm for insets.
Fig 4.
Age-associated changes in AmBRP and Synapsin levels in the central brain of Apis mellifera.
a Representative Western blot of central brains from 1-, 8-, 15-, 29- and 43-day-old bees. Shown are AmBRP proteins migrating around 220 kDa, Synapsin proteins around 70 kDa and the α-Tubulin band around 60 kDa. b-c Results from the quantitative Western blot analysis using central brain homogenate from 1-, 8-, 15-, 29- and 43-day-old bees. b The level of AmBRP in the central brain is increased in the group of 15-day-old worker bees compared with 1-day-old bees. c The level of Synapsin is increased in the group of 15-, 29- and 43-day-old worker bees compared with 1-day-old bees. Box blots show median, 25% and 75% quartiles and value range (min-max). (*) Significant differences (p < 0.05) detected with Mann-Whitney U test after Kruskal Wallis ANOVA.
Fig 5.
Schematic of the pixel counting method.
Schema presenting the method of counting pixels with an antibody signal in regions of interest (ROIs) located in the medial calyces of the honeybee brain. Black squares in the medial calyx (F-actin labeled, scale bar is 20 μm) show the approximately location of these ROIs. Two ROIs were placed in the lip (LI) and two in the dense collar (dCO). One ROI covers an area of 400 μm2 (86 x 86 pixels). The number of pixels that were positive for a specific antibody (i.e. that had an intensity value over a certain threshold, for details see material and methods) were counted.
Fig 6.
The median number of anti-BRPlast200- and anti-SYNORF1-positive pixels per ROI varies with age in lip and collar.
a The median number of anti-BRPlast200-positive (anti-BRP) pixels per ROI in the collar is higher in 43-day-old bees compared with 1-, 8- and 15-day-old bees. b The median number of anti-SYNORF1-positive (anti-SYNORF1) pixels per ROI in the collar is lower in 43-day-old bees compared with 1- and 15-day-old bees. c The ratio of anti-BRPlast200-positive pixels to anti-SYNORF1-positive pixels per ROI in the collar is higher in 43-day-old bees compared with 1-, 8- and 15-day-old bees. d. 1-day- and 43-day-old bees differ in their median number of anti-BRPlast200-positive pixels per ROI in the collar. e The median number of anti-SYNORF1-positive pixels did not change with age in the lip. f The ratio of anti-BRPlast200-positive pixels to anti-SYNORF1-positive pixels per ROI in the lip is higher in 8-day-old bees compared with 1-day-old bees and in 43-day old bees compared with 15- and 29-day-old bees. Box blots show median, 25% and 75% quartiles and value range (min-max). (*) Significant differences (p < 0.05) detected with Mann-Whitney U test after Kruskal Wallis ANOVA.