Coexpression of two visual pigments in a photoreceptor causes an abnormally broad spectral sensitivity in the eye of the butterfly Papilio xuthus

Abstract

The compound eye of the butterfly Papilio xuthus consists of three different types of ommatidia, each containing nine photoreceptor cells (R1-R9). We have found previously that the R5-R8 photoreceptors of type II ommatidia coexpress two different mRNAs, encoding opsins of green- and orange-red-absorbing visual pigments (Kitamoto et al., 1998). Do these cells contain two functionally distinct visual pigments? First, we identified the sensitivity spectrum of these photoreceptors by using combined intracellular recording and dye injection. We thus found that the R5-R8 of type II ommatidia have a characteristic sensitivity spectrum extending over an excessively broad spectral range, from the violet to the red region; the photoreceptors are therefore termed broadband photoreceptors. The spectral shape was interpreted with a computational model for type II ommatidia, containing a UV visual pigment in cells R1 and R2, two green visual pigments in cells R3 and R4, a far-UV-absorbing screening pigment (3-hydroxyretinol) in the distal part of the ommatidium, and a red-screening pigment that surrounds the rhabdom. The modeling suggests that both visual pigments in the R5-R8 photoreceptors participate in phototransduction. This work provides the first compelling evidence that multiple visual pigments participate in phototransduction in single invertebrate photoreceptors.

Figure 1.

Anatomy of the compound eye of P. xuthus; the dorsal side corresponds to the top edge of the micrographs. a, Diagram of three types of ommatidia (from left to right, types I–III). Each ommatidium contains nine photoreceptors, R1–R9 (1–9). R1–R4 are distal photoreceptors, which form the distal two-thirds of the rhabdom; R5–R8 are proximal photoreceptors, forming the proximal one-third of the rhabdom; and R9 is a basal cell. R1 and R2 contain distally purple pigment granules (distal pigment). Types I and II have red pigmentation around the rhabdom, whereas type III has yellow pigmentation (perirhabdomeral pigment). In type II, a fluorescing pigment, 3-hydroxyretinol, is concentrated in the distal portion of the photoreceptor layer (indicated by blue). b, Transverse, unstained section through the proximal tier of the retina, showing red (black arrowhead; type I or II ommatidium) or yellow (white arrowhead; type III ommatidium) pigmentation around the rhabdom. c, UV-induced fluorescence caused by 3-hydroxyretinol marks the type II ommatidia (from eye in intact, living animal). d, e, In situ hybridization of PxL2 (green-absorbing visual pigment) (d) and PxL3 (orange-red-absorbing visual pigment) (e) mRNA in adjacent sections through the proximal tier. R5–R8 are exclusively labeled with either the PxL2 probes (solid circles; type III) or the PxL3 probe (dotted circles; type I). R5–R8 of type II ommatidia are labeled by both PxL2 and PxL3 probes (dashed circles). The table is a summary of the characteristics of the three ommatidial types. In addition to the perirhabdomeral pigmentation and the autofluorescence, it presents the spectral sensitivity of the photoreceptors [S(λ)] and the visual pigment opsins expressed in the ventral half of the eye. V, Violet; B, blue; DG, double-peakedgreen; SG, single-peakedgreen; R, red; BB, broadband. The sensitivity spectrum of R9 photoreceptors is an open question; it probably peaks in the orange wavelength range, but electrophysiological data are lacking so far. For details of S(λ), see Bandai et al. (1992), and for visual pigments, see Kitamoto et al. (1998, 2000). For type I, the spectral type of R1 and R2 may be interchanged (asterisks).

Figure 2.

Measurements of photoreceptor sensitivity spectra. a, Typical recording from a broadband unit. Receptor potentials elicited by a series of equiquantal monochromatic flashes of 20 nm step (from 300 to 700 nm for the first set and from 700 to 300 nm for the second set) and response–stimulus intensity relationships at 560 and 460 nm over a range of 2.5 × 10² ∼ 10⁶ photons·facet^-1·sec^-1 with 0.25 log unit increments are shown. Numbers attached to the arrows indicate the power (i): the intensity of the weakest (i = 2) and strongest (i = 6) pulse thus was 2.5 × 10² and 2.5 × 10⁶ photons·facet^-1·sec^-1, respectively. The V_max and n values of this unit are 46.3 mV and 0.54, respectively. Calibration: 10 mV. b, A set of V–log I functions recorded from a single broadband unit at 420 (filled circle), 440 (open circle), 540 (filled square), and 620 (open square) nm, with the best fits of the Naka–Rushton function. The V_max and n values of this unit at different wavelengths are as follows: 44.5 mV and 0.52 at 420 nm, 43.0 mV and 0.53 at 440 nm, 43.0 mV and 0.52 at 540 nm, and 42.7 mV and 0.55 at 620 nm, respectively. c, Average sensitivity spectrum of the broadband R5–R8 photoreceptors (10 cells; mean ± SE (error bars); filled circles joined by straight lines) and calculated absorption spectra with incident light of different polarization angles: 0^o (dotted curve), 90^o (dashed curve), and 35^o and 125^o (solid curve).

Figure 4.

Calculated (R1 and R2, dotted curve; R3 and R4, dashed curve; R5–R8 at polarization 0°, solid curve) and recorded (violet receptor, open circles; single-peaked green receptor, triangles; broadband receptor, filled circles) sensitivity spectra of the photoreceptor classes in type II ommatidia.

Figure 3.

Lightmicroscopicidentificationofthelocationofabroadbandunit.Thedorsalside of the eye, which was identified by the hole made in the cornea for electrode insertion (data not shown), corresponds to the top edge of all micrographs. The direction allows us to identify the photoreceptor number unambiguously (Fig. 1a). a, The ommatidium containing the Lucifer yellow-injected unit (arrowhead). b, UV excitation showing that the ommatidium was of type II (arrowhead). c, Section of the eye observed under violet excitation. The unit was a proximal photoreceptor R8 (arrowhead). d, Regular transmission microscopy revealed that the ommatidium of the labeled photoreceptor (arrowhead) contained red pigment. Scale bars: a, b, 100 μm; c, d, 10 μm.