Louis, MO, USA). Secondary antibodies (α-mouse

IgG and α-rabbit IgG) conjugated to peroxidase were obtained commercially from Boehringer Mannheim (Mannheim, Germany). Adult honey bees (workers, drones, and queens) were collected from an A. mellifera colony (Africanized hybrids) at the experimental garden of the Federal University of Uberlandia (Uberlândia, MG, Brazil). To distinguish between nurse and forager worker honey bees, physical features, i.e., coat condition and damage to wings were considered, as well as the development of the hypopharyngeal gland observed at the time of brain dissections. Pre-pupal honey bee larvaes were collected from A. mellifera colonies (Africanized hybrids) and maintained at the experimental apiary of the University of São Paulo (Ribeirão Preto, SP, Brazil). Rabbits and rats used in the assay described in Fig. 1 were provided PD0332991 mouse by the University’s Animal Facility and were used under the supervision of the Animal Experiments Review Board at our University. Honey bees were anesthetized on ice and dissected. Larval ganglia and adult brains were removed, frozen in liquid nitrogen, and stored in microtubes at −80 °C. The tissue samples (1 worker/queen or ∼30 worker/drone bee brains, or 2 rabbit/rat GDC-0973 in vitro brains) were homogenized with a hand blender in cold homogenization buffer (40 mM Hepes, pH 7.7, 10 mM EDTA, 2 mM EGTA, 5 mM ATP, 2 mM

DTT, 1 mM benzamidine, 0.1 mM aprotinin and 0.5 mM PMSF). Supernatants were obtained by centrifugation at 40,000g for

40 min at 4 °C. When necessary, protein extracts were concentrated by precipitation with 10% trichloroacetic acid for 15 min on ice, which was followed by centrifugation at 12,000g for 10 min at 4 °C. The precipitates were then solubilized in a small volume of SDS–PAGE sample buffer (100 mM Tris–HCl, pH 8.0, and 25% glycerol). The optical and antennal lobes, mushroom bodies and central region from thirty honey bee brains were dissected, homogenized and centrifuged as described above. Total protein concentrations ( Bradford, 1976) were determined to allow comparison SDS–PAGE and Western blot analyses, as described below. Total protein samples (20 μg) were applied to 5–22% polyacrylamide gradient gels under denaturing conditions (Laemmli and Favre, 1973). next The molecular weight markers were purchased from Sigma–Aldrich (St. Louis, MO, USA), and the gels were stained with Coomassie brilliant blue. For immunoblotting, proteins were transferred to nitrocellulose membranes in Tris–glycine buffer as described by (Towbin et al., 1979). The blots were incubated with 5% dried milk in Tris-buffered saline (TBS-T) (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) and probed with primary antibodies diluted to 0.2 μg/mL in TBS-T and a peroxidase-conjugated anti-rabbit IgG secondary antibody.