Transmission electron microscopy

Pupal cases were removed with fine forceps and the pupae dissected in fixative (2.0% glutaraldehyde in 0.05 M phosphate buffer, pH 6.8; as described by Tilney and Tilney, 1994). The dorsal thorax explant was then transferred to fresh fixative. Pupae were fixed at room temperature for 2-4 h and then transferred to 4°C. After three 10 min washes with 0.05 M phosphate buffer (pH 6.8) on ice, pupae were postfixed with 1% OsO₄ in 0.05 M phosphate buffer (pH 6.8) for 45 min on ice. The fixed pupae were then washed three times for 10 min in water and stained en bloc with 0.5% aqueous uranyl acetate overnight on ice. After washing for 5 min with water, pupae were dehydrated in a graded acetone series (25, 50, 70, 80, 90, 95 and 99.5%) for 15 min at each concentration and transferred to 100% acetone for two 20 min incubations. Polybed 812 (Polysciences) or Quetol 651 (NEM) was used as the resin. When Polybed 812 was used, pupae were incubated in propylene oxide for 20 min twice, infiltrated with a 1:1 mixture of propylene oxide:Polybed 812, and finally placed into 100% Polybed 812. The resin was polymerized for more than 48 h at 60°C. For Quetol 651, pupae were incubated in a 1:1 mixture of n-butyl glycidyl ether (QY1):acetone for 20 min, incubated in 100% QY1 for 20 min, infiltrated with a 1:1 mixture of QY1:Quetol 651 for 2 h, and finally placed into 100% Quetol 651. The resin was polymerized for more than 48 h at 60°C. Semi-thin (0.4-0.7 µm) sections were cut from the anterior side of the pupae and examined using Toluidine Blue staining to confirm orientation. Based on the appearance of the dorsal thoracic microchaetes, the orientation of the pupae was carefully readjusted to obtain a perfect transverse section of the bristle. After confirming orientation, ultrathin sections (∼60 nm) were cut and mounted on 50-mesh Formvar-coated copper grids. The sections were stained with 4% aqueous uranyl acetate in the dark for 10 min, stained with Reynold's lead citrate for 3 min, and then coated with a thin layer of carbon. Observations were performed using a JEM-1010 transmission electron microscope (JEOL) at 100 kV accelerating voltage, and images were captured by a Gatan Bioscan 792 digital camera.