TUNEL and DAPI staining of cross sections in pupal wings.

<p>TUNEL assays (green: A, C, E, G) and DAPI staining (cyan: B, D, F, H) of cross sections of female and male pupal wings. (A) Cross section of female pupal wing on Day 4 after the beginning of pupal-adult development. (C) Cross section of female pupal wing on Day 6 after injection of 5.4 µg 20E. (E) Cross section of male pupal wing on Day 7 after beginning of pupal-adult development. (G) Cross section of male pupal wing on Day 8 after injection of 5.4 µg 20E. Note that TUNEL signals (light green) indicated by white arrows were visible in areas of the female wing epithelium. Scale bar  = 50 µm.</p

Developmental profiles of pupal wings in <i>Nyssiodes lefuarius</i>.

<p>Pupal wings of females (A–D) and males (E–H). (A–D) modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089435#pone.0089435-Niitsu1" target="_blank">[12]</a>. In non-differentiation, the pattern of the pupal wing trachea in the female (A) is the same as that of the male (E) during the non-differentiation period. After differentiation, the pupal wing of females is degenerated. Note the significant pupal-wing degeneration on Day 3 (B), Day 4 (C) and Day 7 (D) after the beginning of pupal-adult development. By contrast, the pupal wing of males is formed normally on Day 10 (F) and Day 20 (G) after the beginning of pupal-adult development. (H) is just before adult eclosion. The arrow (A–D) points to the distal end of the degenerating wing of female. White arrowheads (F, G) indicate the position of the bordering lacuna (BL). Scale bar  = 1 mm.</p

Whole mount images and cross sections of female pupal wings.

<p>Day 2 (A, D), Day 3 (B, E), and Day 4 (C, F) after the beginning of adult development. (A–C) Whole mount of female-pupal wings dissected from a pupa. (D–F) Cross sections of female-pupal wings. a-a′, b-b′, and c-c′ levels of the sections depicted in D–F. The box area in (F) corresponds to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089435#pone-0089435-g003" target="_blank">Fig. 3(B)</a>. An epithelial fragment was degenerating into the wing lumen (B, double arrowheads). An apoptotic body-like structure was also visible (F, black arrows). Note that the position of the epithelial nuclei shrank gradually (D–F, arrowheads). BL, basal lamina; PL, plasmatocytes; GR, granulocytes. Scale bar  = 20 µm.</p

Morphological changes in pupal wings induced by injection of ecdysteroid (20-hydroxyecdysone, 20E).

<p>(A) Female-pupal wing without application of 20E on Day 6 after injection of 6 µl (4.7 µg) ethanol. Female-pupal wing on Day 6 (B), Day 8 (C), and Day 11 (D) after injection of 5.4 µg 20E. (E) Male-pupal wing after injection of 6 µl (4.7 µg) ethanol. Male-pupal wing on Day 6 (F), Day 8 (G), and Day 11 (H) after injection of 5.4 µg 20E. All wing epithelia are attached to their pupal-wing case. Arrows point the distal end of the degenerating wing epithelium in the female (B–D). Scale bar  = 1 mm.</p

Cross sections of the pupal wing epithelium induced by injection of 20E.

<p>The pupal-wing epithelium of females (A–D) and males (E–H). Pupal wing epithelium of females (A) and males (E) on Day 6 after injection of 6 µl ethanol. Pupal-wing epithelium of females on Day 6 (B), Day 8 (C) and Day 11 (D) after injection of 5.4 µg 20E. Phagocytes (white arrows) and apoptotic-body-like structures (black arrows) are visible (D). Pupal-wing epithelium of males on Day 6 (F), Day 8 (G) and Day 11 (H) after injection of 5.4 µg 20E. Scale is visible in the pupal epithelium (F, G, H). BL, basal lamina; P, phagocyte; S, scale; T, trachea; t, tracheole. Scale bar  = 20 µm.</p

Transmission electron microscope micrographs of the pupal wing epithelium of females.

<p>Cell death of the pupal wing epithelium of females occurred strikingly on Day 4 after the beginning of pupal-adult development. (A) Phagocyte (black arrow) is engulfing fragmented forms of dead cells and lysosomes. (B) Condensed chromatin (open arrows) derived from nuclei are visible. (C) Autophagosome fuses to lysosomes to become an autolysosome. (D) Condensed chromatin and endoplasmic reticulum are visible. N, normal nuclei; ER, endoplasmic reticulum; BL, basal lamina; L, lysosome; AP, autophagosome; AV, autophagic vacuole. Scale bars: 5 µm for A–B and 2 µm for C–D.</p

Induction ratio of 20E application by injection on wing morphogenesis at Day 14 in <i>Nyssiodes lefuarius</i>.

<p>Induction ratio of 20E application by injection on wing morphogenesis at Day 14 in <i>Nyssiodes lefuarius</i>.</p