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First published online March 22, 2004
Journal of Experimental Biology 207, 1479-1486 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00913

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Immunohistochemical localization of Papilio RBP in the eye of butterflies

Motohiro Wakakuwa¹, Koichi Ozaki² and Kentaro Arikawa^1,*

¹ Graduate School of Integrated Science, Yokohama City University, Yokohama, Kanagawa 236-0027, Japan
² Graduate School of Frontier Bioscience, Osaka University, Toyonaka, Osaka 560-0043, Japan

View larger version (51K): [in a new window]   Fig. 1. SDS-PAGE (lanes 1, 2) and immunoblot (lanes 1', 2') analyses of the soluble fraction of the Papilio compound eye homogenate (lanes 1, 1') and purified Papilio RBP (lanes 2, 2'). Anti-Papilio RBP antiserum monospecifically detected Papilio RBP at about 31 kDa.

View larger version (138K): [in a new window]   Fig. 2. Light microscopic immunohistochemistry using the anti-Papilio RBP in the retina of Papilio xuthus. (A) Schematic drawing of an ommatidium. Longitudinal view. Arrows on the left indicate the approximate locations where sections B–D were obtained. BM, basement membrane; C, corneal facet lens; CC, crystalline cone; PPC, primary pigment cell; Pr, photoreceptor; Rh, rhabdom; SPC, secondary pigment cell; TC, tracheal cell. (B) Cross section through the distal portion of the retina. The pigment cells (black arrowheads) were labeled. Photoreceptors (white arrowheads) were not labeled. Non-immune serum gave no labeling (inset). (C) Transverse section through the proximal part of the retina. The secondary pigment cells (black arrowheads) were labeled, but no photoreceptors were labeled. (D) Longitudinal section around the basement membrane (BM). The tracheal cells (white arrowheads) as well as the secondary pigment cells (black arrowheads) were strongly labeled. Scale bars, 30 µm.

View larger version (134K): [in a new window]   Fig. 3. Electron miscroscopic immunohistochemistry using anti-Papilio RBP secondary antibody conjugated to 15 nm colloidal gold. The areas marked by broken squares in A, C, E, G and I are magnified in B, D, F, H and J, respectively. (A,B) Primary pigment cells (PPC) surround the crystalline cone (CC). Both the cytoplasm and the nucleus (N) were densely labeled. (C,D) Photoreceptor cells (Pr). No labeling was found in the cell body of the photoreceptor or in the rhabdom (Rh). (E,F) Secondary pigment cells (SPC) fill the space between photoreceptors (Pr). The cytoplasm of the secondary pigment cell was strongly labeled. (G,H) The nucleus (N) of the secondary pigment cell was also strongly labeled. (I,J) Tracheal cells. Labeling was found both in the cytoplasm surrounding the tracheole (T) and in the nucleus (N). Photoreceptor axons (Ax) were not labeled. Scale bars, 2 µm (A,I); 1 µm (B,C,E,G,J); 500 nm (D,F,H).

View larger version (14K): [in a new window]   Fig. 4. Comparison of the labeling density quantified by particle density in the electron miscroscopic immunohistochemistry. Values are means ± S.E.M. of the particle density in nine separate regions. *Statistically significant difference (P<0.01, one-way ANOVA, Tukey test). For abbreviations, see Fig. 2. Cy, cytoplasm; Nu, nucleus.

View larger version (74K): [in a new window]   Fig. 5. Native PAGE of crude retinal extract of lepidopteran species belonging to the superfamily Papilionoidea. Fluorescence under UV light (left); Coomassie Brilliant Blue (CBB) staining (right). Lane 1, Papilio xuthus (Papilionidae); 2, Papilio machaon (Papilionidae); 3 Papilio protenor (Papilionidae); 4, Papilio helenus (Papilionidae); 5, Graphium sarpedon (Papilionidae); 6, Vanessa indica (Nymphalidae); 7, Pieris rapae (Pieridae).

View larger version (26K): [in a new window]   Fig. 6. SDS-PAGE (left) and immunoblot (right) analyses using purified fluorescing proteins from the native gel shown in Fig. 5. Lane 1, Papilio xuthus; 2, Papilio machaon; 3, Papilio protenor; 4, Papilio helenus; 5, Graphium sarpedon; 6, Vanessa indica; 7, Pieris rapae. Except for Vanessa indica, the purified fluorescing proteins appeared as single bands. The molecular masses of these proteins were between 25 kDa and 31 kDa, all of which were detected by the anti-Papilio RBP. In Vanessa indica, only the 25 kDa protein was detected by anti-Papilio RBP.

View larger version (59K): [in a new window]   Fig. 7. Native PAGE (left), SDS-PAGE (middle) and immunoblot (right) analyses of the crude retinal extracts of various insect species. Lane 1, Papilio xuthus as the control; 2, Parnara guttata (Hesperidae, Lepidoptera); 3, Orthetrum albistylum speciosum (Libellulidae, Odonata); 4, Graptopsaltria nigrofuscata (Cicadidae, Hemiptera); 5, Oedaleus infemalis (Acrididae, Orthoptera). No proteins from insects other than Papilio xuthus cross-reacted with the anti-Papilio RBP.

View larger version (113K): [in a new window]   Fig. 8. Light microscopic immunohistochemistry using the anti-Papilio RBP in the retina of Peris rapae. (A) Transverse section of distal region of the retina. Both primary pigment cells (small arrowheads) and secondary pigment cells (arrowheads) were labeled. Photoreceptors (white arrowhead) were not labeled. Non-immune serum gave no labeling (inset). (B) Transverse section of the proximal region of the retina. The secondary pigment cells (black arrowheads) were labeled, but the photoreceptors were not (white arrowheads). (C) Longitudinal section around the basement membrane (BM). The tracheal cells (white arrowheads) were strongly labeled. Scale bars, 30 µm.