Pupae were either treated topically on the abdominal cuticle with 2 µl of a juvenile hormone-III solution (Sigma, 5 mg/ml in acetone), or received an injection of 1 µl ecdysone solution (Sigma, 5mg/ml in ethanol/Ringer solution 1:4). Control experiments were performed with the respective solvents. At least 3 groups consisting of 3–6 individuals each (for a total of 9–18 bees) were used in each experiment. In studies on transcriptional regulation, actinomycin D (10 µg in DMSO/Ringer's solution 1:1) was injected 1h prior to juvenile hormone application. All treatments were performed at one developmental stage prior to the initiation of hemolymph sampling at the next stage, e.g., when juvenile hormone was applied at the Pbm I-stage, the hemolymph sampling started with Pbm II.
Hemolymph was obtained from pupae from a small incision in the dorsal cuticle between the 2nd and 3rd tergite, or, in adults by cutting off the wings at their bases. After adding a few crystals of phenylthiourea to prevent melanization, the hemolymph pools were centrifuged (3000g for 10 min at 4°C) and the supernatants were stored at −20°C.
Hemolymph was obtained from pupae from a small incision in the dorsal cuticle between the 2nd and 3rd tergite, or, in adults by cutting off the wings at their bases. After adding a few crystals of phenylthiourea to prevent melanization, the hemolymph pools were centrifuged (3000g for 10 min at 4°C) and the supernatants were stored at −20°C.
