3.5.2. Measuring the rate of JH biosynthesis
1. Remove the paired corpora allata (CA) from the base of the bee brain (see the section on isolation of the retrocerebral complex in the BEEBOOK paper on anatomy and dissection (Carreck et al., 2013). While minimizing the amount of brain tissue attached to the glands, leave any attached tracheal elements as these enhance buoyancy. This will help ensure the glands stay near the surface of the medium during incubation, enhancing oxygenation and tissue activity (Kaatz et al., 1985; Holbrook et al., 1997).
2. Separately preincubate each dissected pair of CA in 100 μl of nonradiolabelled Grace’s medium for 15 min in borosilicate glass culture tubes (6 x 50 mm) at room temperature.
3. Aliquot 100 μl of radiolabelled medium into a duplicate set of labelled glass culture tubes.
4. Using a small copper wire hoop, pick up a droplet of medium containing the preincubated CA and transfer it to the corresponding tube containing the radiolabelled medium.
Ensure that the tissue stays close to the surface to enhance oxygenation.
5. Prepare a negative control tube containing radiolabelled media with no added tissue.
6. Loosely cover the tubes with plastic wrap to prevent desiccation, but ensure an adequate supply of oxygen.
7. Incubate the samples on a variable-plane mixer set at a 15°-17° angle at 90 rpm at 27°C for 3h.
The functional capacity of the A. mellifera CA begins to degrade within a few hours after dissection, so the 3h incubation allows for an accurate assessment of the rate of release and maximizes the amount of JH available for detection purposes.
8. At the end of the incubation period, remove the CA.
9. Add 250 μl of chilled isooctane to each tube.
10. Incubate on the variable-plane mixer for an additional 15 min.
11. Vortex the samples for 1 min.
12. Centrifuge at 10,000 x g for 10 min.
13. Using a pipettor with a gel loading tip, transfer 100 μl of the upper isooctane layer to each of two labelled scintillation vials so that each extract is assayed in duplicate.
14. Reduce the volume of the isooctane to near-dryness under nitrogen or in a vacuum concentrator.
15. Add scintillation fluid (see section 3.5.1., step 7.2) to the vial.
16. Vortex for 1 min.
17. Let rest for 1 h before measuring the radioactivity in a scintillation counter.