Fig. 3. Confocal images of the larval epiproctodeal gland (A,B), axons in the proctodeal nerve (C) and median neuroendocrine cells in the adult brain of Manduca sexta (D,E). (A) Myoinhibitory peptide (MIP)-immunostaining of a whole mount of the epiproctodeal gland (EpG) and its neurohemal processes on the proctodeal nerve (PN) of a third-instar larva. In addition, MIP-immunoreactive (MIP-IR) axons that extend from neurons in the terminal ganglion and terminal nerve (TN) can be seen in the proctodeal nerve (arrow). (B) Acridine Orange staining of a cell in a whole mount of an epiproctodeal gland of a fourth-instar larva at a late stage of spiracular apolysis. The stain demonstrates a high concentration of RNA in the cytoplasm at this stage; the unstained spherical structures in the cell have been shown in previous studies to be nuclei. (C) Whole mount of a Biocytin-stained, proctodeal nerve filled from the base of the terminal nerve of a fourth-instar larva. The location of one of the gland cells (EpG) can be distinguished by its weak background staining. Arrows indicate the direction of filling with Biocytin. Note that branches of these axons do not terminate on the epiproctodeal gland, and, therefore, the gland apparently is not innervated. Moreover, the staining failed to demonstrate any processes extending from the gland to the terminal abdominal ganglion. (D) Double-immunostaining of MIP-IR (red) in the 1A₄ cells and of small cardioactive peptide B (SCP_B)-IR (green) in the 1A₅ median neuroendocrine cells. Vibratome section (200 µm), frontal plane of the adult brain. (E) Double-immunostaining for MIP (red) and allatostatin (green), showing that immunoreactivity to these two peptides is co-localized (yellow) in the 1A₄ cells. Vibratome section (200 µm), frontal plane of the adult brain. Scale bars, 400 µm (A) and 100 µm (B–E).

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