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First published online February 4, 2005
Journal of Experimental Biology 208, 687-696 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01453

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Eyeshine and spectral tuning of long wavelength-sensitive rhodopsins: no evidence for red-sensitive photoreceptors among five Nymphalini butterfly species

Adriana D. Briscoe^1,* and Gary D. Bernard²

¹ Comparative and Evolutionary Physiology Group, Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, USA
² Department of Electrical Engineering, University of Washington, Seattle, WA 98195-2500, USA

View larger version (21K): [in a new window]   Fig. 1. Diagram of the epi-microspectrophotometric apparatus. The monochromatic beam contains: Qm, Ealing 150 W optically stabilized Xenon-arc illuminator; Fs, heat filter and stray light filter; G, computer-controlled monochromator with entrance slit An and exit slit Ax; Lm, quartz condenser; Af, field aperture; W, computer-controlled counter-rotating neutral density wheels; Sm, computer-controlled Uniblitz shutter; M, mirror. The adapting beam contains: Qa, 45 Wquartz-iodine illuminator; Fh, heat filter; Fd, neutral filter; Fc, cut-off filter; La, telephoto lens; Aa, field aperture; Sa, Uniblitz shutter. The two collimated beams are combined in quartz beam-splitter Bq and inserted into the Leitz Pol-Opak epi-illuminator equipped with 620-257 illumination slide designed for low stray-light. A, aperture diaphragm; Lo, microscope objective, either Leitz 8X/0.18P acromat/215 or Zeiss Luminar-25 mm/0.15; Le, GF10X eye piece; E, eye of completely intact butterfly mounted on Leitz UT-4 goniometric stage. The Leitz UV-MPV photometer head contains Ap, adjustable/centrable photometer diaphragm; Fp, stimulus-blocking filter; P, cooled Hammamatsu R928 photomultiplier.

View larger version (24K): [in a new window]   Fig. 3. (A) Log-reflectance spectra of eyeshine measured from a medio-ventral region of the dark-adapted eyes of Vanessa cardui (green filled circles) and Pieris rapae (red filled circles). The open circles are spectra following substantial photo-conversion of LW rhodopsin to blue-shifted metarhodopsin, for Vanessa (green open circles) and Pieris (red open circles). (B) Log-reflectance spectra of eyeshine measured from a medio-ventral region of the dark-adapted eyes of nymphalid butterflies Vanessa cardui (green filled circles), Nymphalis antiopa (black filled circles), Inachis io (orange filled squares), Siproeta stelenes (cyan filled circles), and Junonia coenia (blue filled circles).

View larger version (113K): [in a new window]   Fig. 2. Eyeshine from Nymphalini butterfly compound eyes. Epi-illumination of a dark-adapted eye showing light reflected from ommatidia viewing the incident illumination. (A) Junonia coenia The whitish appearance of the eye is likely due to light reflecting screening pigments located distally within the primary pigment cells (see Yagi and Koyama, 1963; Stavenga, 2002a) (B) Vanessa cardui. (C) Nymphalis antiopa. (D) Siproeta stelenes.

View larger version (31K): [in a new window]   Fig. 4. Absorbance spectra derived from partial bleaches of long wavelength rhodopsins in eyes of completely intact butterflies. The solid curves are idealized spectra based upon the A1-SSH template (Stavenga et al., 1993). (A) R522 of Siproeta stelenes; dorsal retina, filled dots and unfilled dots ventral retina of the same animal. (B) R510 of Junonia coenia, from the medio-ventral retina. (C) R530 of Inachis io, from the ventral retina just below the acute zone. (D) R534 of Nymphalis antiopa, from the medio-equatorial retina. Absorbance units are one-way, base-10 densities of rhabdom content, averaged over all illuminated ommatidia.

View larger version (40K): [in a new window]   Fig. 5. Alignment of the deduced amino acid sequences of the nymphalid LW opsin. Blocks indicate the location of the transmembrane domains (I-VII) within the opsin protein sequence. Asterisks indicate candidate spectral tuning sites of known spectral shifts among vertebrate rhodopsins.

View larger version (25K): [in a new window]   Fig. 6. Phylogeny of butterfly opsins. The neighbor-joining tree (shown) was constructed using a first plus second nucleotide sites and Tamura-Nei correction. Bootstrap support values (out of 500 replicates) for the neighbor-joining tree are shown above the nodes and bootstrap support (out of 100 replicates) for the stepmatrix-weighted maximum parsimony tree are below. References for additional max values are given in Arikawa et al. (1999) and Briscoe (2001). GenBank accession numbers are: Heliconius sara (OPS1, AF126753; Hsu et al., 2001), Manduca sexta (1, L78080; 2, L780801; 3, AD001674; Chase et al., 1997), Papilio glaucus (PglRh1, AF077189; PglRh2, AF077190; PglRh3, AF067080; PglRh4, AF077193; PglRh5, AF077191; PglRh6, AF077192; Briscoe, 2000), Papilio xuthus (PxRh1, AB007423; PxRh2, AB007424; PxRh3, AB007425; Kitamoto et al., 1998), Galleria mellonella (AF385330), Precis coenia (AF385332), Spodoptera exigua (AF385331), Vanessa cardui (AF385333; Briscoe, 2001), Bombyx mori (AB064496; Shimizu et al., 2001).