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First published online August 25, 2003

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The retina of Manduca sexta: rhodopsin expression, the mosaic of green-, blue- and UV-sensitive photoreceptors, and regional specialization

Richard H. White^1,*, Huihong Xu², Thomas A. Münch³, Ruth R. Bennett¹ and Erin A. Grable¹

¹ Department of Biology, University of Massachusetts Boston, 100 Morrissey Blvd, Boston, MA 02125-3393, USA,
² MGH Cancer Center, Harvard Medical School, 13th Street, Charlestown, MA 02129, USA
³ Helen Wills Neuroscience Institute, University of California Berkeley, 145 Life Sciences Addition, Berkeley, CA 94720, USA

View larger version (141K): [in a new window]   Fig. 1. Illustration for method of measuring rhabdomere volumes. (A) Profiles (see Fig. 5A) of two adjacent rows of retinulae traced from electron micrographs of a tangential section extending from the retinal surface to the basement membrane. (B) Inferred plane of section projected across a light micrograph of a longitudinally sectioned retina.

View larger version (69K): [in a new window]   Fig. 2. Retina of Manduca sexta. (A) Dorsal aspect of an unfixed retina dissected free of the cornea and clear zone. Anterior is to the right. The blue patch is the dorsal rim area. Scale bar, 0.5 mm. (B) Freehand vertical section of a glutaraldehyde-fixed retina photographed in reflected light. Dorsal is at the top, distal to the left. The highly reflective tracheoles (t) extend almost to the distal surface (ds) of the retina, except in the dorsal rim area (dr). Scale bar, 100 µm. (C) Retinulae dissected from the center of a fixed retina photographed in transmitted light: tracheoles are opaque, extending across the retina to just below the distal tips of the retinulae. bm, basement membrane beneath the retina. Scale bar, 10 µm. (D) Dorsal rim retinulae. Tracheoles envelop only the proximal ends of the retinulae. Scale bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 3. Rhabdomere volumes of receptor cells near the center of the retina. (A) Light micrograph of longitudinal section showing two retinulae. bm, basement membrane; ne, nuclei of elongate photoreceptors; np, nucleus of small proximal receptor; r, rhabdom; t, tracheoles; te, tracheole ends. (B) Rhabdomere volumes, calculated from 21 virtual sections (see Materials and methods and Fig. 1A) of photoreceptors in relation to depth of retina. Each rectangle represents a virtual section: its vertical side represents the section thickness; the horizontal side represents the area of the rhabdomere. The calculated rhabdomere volume for each cell type is indicated.

View larger version (108K): [in a new window]   Fig. 4. Comparison of dorsal rim retinulae with nearby retinulae of the yellow retina in the same section stained with anti-P357 and photographed with phase interference optics to enhance images of rhabdoms. (A) Dorsal rim retinulae with typical butterfly-shaped rhabdoms. Note the absence of surrounding tracheoles. The narrow ventral dv cell is stained in the retinula on the far right. (B) Retinulae surrounded by tracheoles (t) from an adjacent region of the yellow retina. Two dv cells are stained in the retinula on the far left; only one cell is stained in the other retinulae. Scale bar, 1 µm.

View larger version (25K): [in a new window]   Fig. 5. Organization of retinulae from the yellow retina and dorsal rim. (A) Tracings of electron micrographs from sectioned retinulae near the center of the retina, showing the component photoreceptors and their rhabdomeres (shaded). The dorsal-ventral axis of the retina is vertical. The depth of each retinular profile from the surface of the retina (see Fig. 3) is indicated. dv, dorsal-ventral receptor cells; ap, anterior-posterior cells; ob, oblique cells; pr, proximal cell. (B) Tracing from electron micrograph of a retinula from the dorsal rim area. The structures of the eight elongate receptor cells do not vary with retinal depth (the basal pr cell has not yet been found). Only the ventral dv cell is present. Orientations of photoreceptor microvilli are indicated by double-headed arrows. Scale bar, 1 µm.

View larger version (112K): [in a new window]   Fig. 6. Proteins from Manduca retina. (A) SDS-PAGE electrophoresis of aliquot parts of retinal homogenate equivalent to 0.1 retina, calibrated with molecular masses of Bio-Rad protein standards. (B,C,D) Western blots with antisera against P520, P450 and P357, respectively, showing a major band at approximately 37 kDa. (E) Control blot lacking primary antibodies.

View larger version (165K): [in a new window]   Fig. 7. Longitudinal sections from the same retina immunostained with antisera to the three characterized Manduca opsins. (A) Anti-P520. (B) Anti-P450. (C) Anti-P357. The dark blotches just above the basement membrane (bm) in A are screening pigment, not stain. Scale bar, 10 µm.

View larger version (138K): [in a new window]   Fig. 8. Adjacent immunostained sections from the ventral domain of the retina, approximately 70 µm from the surface of the retina. The dorsal-ventral retinal axis is vertical. (A) Anti-P520. Rhabdomeres of ap and ob cells are stained. (B) Anti-P357. Some dv cells are stained. A retinula in which both dv cells are stained is marked with an asterisk. Scale bar, 10 µm.

View larger version (134K): [in a new window]   Fig. 9. Adjacent immunostained sections from the ventral domain of the retina. (A) Anti-P357. (B) Anti P-450. Asterisks mark retinulae in which one dv cell stains for P357, the other for P450. Scale bar, 10 µm.

View larger version (82K): [in a new window]   Fig. 10. Retinal mosaic of blue- and UV-sensitive photoreceptors. (A) Diagram of the Manduca retina drawn to scale, showing locations of the retinal patches illustrated in B-E. Anterior is to the right. The equatorial division (eq) between dorsal and ventral retinal domains is indicated, as is the dorsal rim area (dr). Scale bar, 0.5 mm. (B,C,E) Diagrammatic representations of retinal patches mapped from adjacent sections stained for P450 and P357, respectively. Each hexagon represents a retinula containing potentially two dv cells. A uniformly colored hexagon represents a retinula containing two identical cells. A hexagon divided in half horizontally is a retinula with unlike or missing cells. Blue represents cells expressing P450; red represents cells expressing P357; white indicates no staining by either antiserum in that position. (B) Domain in the dorsal retina that extends into dorsal rim region (dr), whose margin is represented by the heavy line. Some dorsal-ventral rows of retinulae terminate before reaching the edge of the retina. Red lines in the dorsal rim region represent the narrow dv cells that express P357. (C) Domain in the ventral retina. (D) Large patch at the center of the retina for which grayscale micrographs of adjacent sections immunostained for P450 and P357 have been superimposed and arbitrarily colored (blue, anti-P450; red, anti-P357) in Adobe Photoshop. Scale bar, 100 µm. (E) Equatorial sector from the adjacent sections shown in D.