Fig. 2. Double wholemount immunohistochemical staining of the epitracheal gland during the fifth instar of Manduca sexta larvae. Only Inka cells (large arrow) were double-labelled by the pre-ecdysis-triggering hormone (PETH) and horseradish peroxidase (HRP) antisera (orange-red colour), while exocrine cells (large arrowhead) reacted just with the FITC-labelled HRP antiserum (yellowish-green colour). The ‘narrow’ (small arrow) and ‘canal’ (small arrowheads) cells plus tracheal cells were revealed by nuclear DAPI dye (blue colour). (A) Strong PETH-immunoreactivity (IR) was detected in Inka cells 8 h before ecdysis into the fifth instar; (B) this staining disappeared completely 5 min after ecdysis. (C,D) Weak PETH-IR was again detected in feeding larvae on day 1 and increased on day 3. (E,F) The Inka cells of wandering larvae (days 5, 6) subsequently showed stronger staining as the entire glands increased in size. (G–I). The epitracheal glands reached maximal size 1–2 days prior to pupal ecdysis (days 8–9). Note that the duct process was quite long and obvious in the feeding and wandering stages (C–F), but was reduced in size 1–2 days prior to ecdysis (A,G–I). (G) A rare case of two Inka cells with one set of gland cells attached to the same trachea. Another separated set of gland cells was attached to the opposite side of this trachea. Days 1–9 refer to days after ecdysis into the fifth instar. Scale bar, 100 µm.