Spectral organization of the eye of a butterfly, Papilio
This review outlines our recent studies on the spectral organization of butterfly compound eyes, with emphasis on the Japanese yellow swallowtail butterfly, Papilio xuthus, which is the most extensively studied species. Papilio has color vision when searching for nectar among flowers, and their compound eyes are furnished with six distinct classes of spectral receptors (UV, violet, blue, green, red, broadband). The compound eyes consist of many ommatidia, each containing nine photoreceptor cells. How are the six classes of spectral receptors arranged in the ommatidia? By studying their electrophysiology, histology, and molecular biology, it was found that the Papilio ommatidia can be divided into three types according to the combination of spectral receptors they contain. Different types of ommatidia are distributed randomly over the retina. Histologically, the heterogeneity appeared to be related to red or yellow pigmentation around the rhabdom. A subset of red-pigmented ommatidia contains 3-hydroxyretinol in the distal portion, fluorescing under UV epi-illumination. The red, yellow and fluorescing pigments all play crucial roles in determining the spectral sensitivities of receptors. Spectral heterogeneity and random array of ommatidia have also been found in other lepidopteran species. Similarities and differences between species are also discussed.
KeywordsColor vision Compound eye Photoreceptor Retina Rhabdom
Spectral receptors in butterfly eyes
When searching for nectar, butterflies find their sources, the flowers, by using their capacity for color vision (Kelber and Pfaff 1999; Kinoshita and Arikawa 2000; Kinoshita et al. 1999) in a way similar to that of honeybees (von Frisch 1914). The color information is, of course, detected by the compound eyes. The human color vision system is trichromatic, and is based on the short-, middle- and long-wavelength cone photoreceptor cells in the retina. Honeybees also have a trichromatic system with three types of spectral receptors in the eye, each peaking in the ultraviolet (UV), blue and green wavelength regions (Menzel and Backhaus 1989). As predicted from the spectral receptor types, honeybees are highly sensitive in the UV, but their sensitivity to red light is weak (von Frisch 1914); in fact, red flowers are not particularly attractive to honeybees. However, a number of butterfly species frequently visit red flowers. This observation raises several questions: (1) do butterflies recognize red?; (2)what type of spectral receptors do they possess?; and (3) how many types are there?
We determined the anatomical identity of the spectral receptors by a conventional method of dye injection: we injected lucifer yellow into a penetrated photoreceptor after recording its spectral sensitivity, and then identified the location of the marked cell by histology. We also used a method we call the polarization method. Photoreceptors in the Papilio ommatidia bear parallel microvilli, making the photoreceptors polarization sensitive (Moody and Parriss 1961). For example, the microvilli of the Papilio R1 and R2 are oriented parallel to the dorso-ventral axis of the eye (see Fig. 1), and therefore these photoreceptors are maximally sensitive to polarized light with e-vector angle parallel to the dorso-ventral axis. By knowing the anatomical organization of the microvilli of the different photoreceptors we thus can determine the identity of a photoreceptor from its polarization sensitivity. After recording the spectral sensitivity, we subsequently recorded the polarization sensitivity to localize the penetrated photoreceptor in the ommatidium. Suppose that the polarization sensitivity of a UV receptor peaks at the angle parallel to the dorso-ventral axis: it means that the UV receptor is either an R1 or an R2. Of course, the polarization method is not applicable to eyes whose rhabdoms are twisted, as in bees and ants (Menzel and Blakers 1975).
The electrophysiological results revealed that the anatomical receptor types do not always have unique spectral properties (summarized in Fig. 1). Note for instance that R1 and R2 are either UV, violet, or blue receptors. Assigning three spectral receptors to only two anatomically distinct photoreceptors means that all ommatidia cannot be identical to each other in terms of the combination of the spectral receptors contained. In other words, the ommatidia must be spectrally heterogeneous.
Colors of ommatidia
What is the function of the pigments? The rhabdom, whose diameter is about 2.5 μm, acts as an optical waveguide because of its slenderness (Nilsson et al. 1988; van Hateren 1989). The red and yellow pigments, positioned within 1 μm of the rhabdom boundary, will absorb light propagated in the boundary wave, and thus function as spectral filters. The filtering effect can be directly observed in a simple experiment in which a fresh compound eye, sliced through the proximal tier, is illuminated from the cut surface and observed from the corneal side. The ommatidia then appear either yellow or (more or less saturated) red (Fig. 3c), corresponding to the color of the pigments surrounding the rhabdom. The color of the purple pigment of R1 and R2 is not observed this way, as it probably serves as a light-controlling, pupillary pigment (Stavenga 1979), although experiments to prove this have not been reported so far.
The less saturated red or whitish transmitting ommatidia (Fig. 3c) are a variant of the deeply red-pigmented ommatidia containing a low amount of red pigment. Most interestingly, these ommatidia emit a strong, whitish fluorescence under UV epi-illumination (Fig. 3d). The fluorescing material is 3-hydroxyretinol concentrated in the distal portion of the fluorescing ommatidia (Arikawa et al. 1999a). About 25% of the ommatidia in the ventral half are of the fluorescing type. The ommatidial fluorescence is not observed in the dorsal half, because there the cornea itself fluoresces, meaning that the ommatidial fluorescence is either absent or obscured by the corneal fluorescence (Fig. 3e). (The border between the fluorescing and non-fluorescing cornea roughly corresponds to the equator of the visual field, which is why we refer to these regions as the dorsal ‘half’ and the ventral ‘half’.)
Taken together, the Papilio compound eye contains at least three types of ommatidia (Fig. 3f), which are deep red-pigmented (type I), red-pigmented and fluorescing (type II), and yellow-pigmented (type III). The three types of ommatidia distribute rather randomly, at least locally in the ventral half of the eye.
Differential expression of visual pigment opsins among the ommatidial types
The probes for the mRNAs of PxUV and PxB exclusively labeled the R1 and R2, which are distal photoreceptors with vertically oriented microvilli (Fig. 5a, b). In type I ommatidia one of the R1 and R2 photoreceptors is labeled with the PxUV probe and the other with the PxB probe: when R1 is PxUV positive, R2 is PxB positive, and vice versa. R1 and R2 of type II are both labeled with the PxUV probe, and those of type III are labeled with the PxB probe. These results match the electrophysiological results: R1 and R2 are short wavelength receptors (Fig. 1).
The PxL1 probe labeled the horizontally oriented distal photoreceptors R3 and R4 only in the ventral half of the retina (Fig. 5e, f). The PxL1 is probably the opsin of a green-absorbing visual pigment, since these photoreceptors are green sensitive (Fig. 1).
The PxL2 probe labeled R3 and R4 in all ommatidia in both the dorsal and ventral halves (Fig. 5g, h). As R3 and R4 are green sensitive, PxL2 must also be an opsin of a green-absorbing visual pigment. The labeling of R3 and R4 in the ventral half by both PxL1 and PxL2 mRNAs suggest the hypothesis that these photoreceptors coexpress both PxL1 and PxL2 visual pigments (Fig. 5f, h).
The labeling pattern of PxL2 and PxL3 in the proximal R5–8 photoreceptors appears to be even more complicated. R5–8 in the type I ommatidia were labeled exclusively with the PxL3 probe (Fig. 5d), whereas those of type III were exclusively labeled with the PxL2 probe (Fig. 5c). Interestingly, R5–8 of type II ommatidia, which fluoresce under UV (Fig. 5i), were double labeled by both the PxL2 and PxL3 probes (Fig. 5c, d).
Spectral tuning and the ommatidial types
The spectral sensitivities of Papilio photoreceptors (Fig. 2) cannot be simply explained by the absorption spectra of visual pigments (Govardovskii et al. 2000). For example, the sensitivity of violet receptors, peaking at 400 nm, is much too narrow compared to the predicted spectrum of a visual pigment absorbing maximally at 400 nm (see Fig. 8). Furthermore, if the colocalized multiple visual pigment mRNAs express functional visual pigments, this should result in characteristically modified spectral sensitivities. We studied the spectral tuning of photoreceptors in detail by postulating the optical interaction of the visual pigments with the red, yellow, and fluorescing pigments. The present understanding of the photoreceptor spectral tuning is reviewed below, with the focus on the red, violet, single-peaked green, and broadband receptors of Papilio.
Summary of the characteristics of the three ommatidial types in Papilio xuthus
Type of ommatidia
Single-peaked green receptors
The UV absorbing function of 3-hydroxyretinol of course affects the spectral sensitivities of R3–8 photoreceptors in type II ommatidia. R3 and R4 are green sensitive in all ommatidia, which is confirmed by both electrophysiology (Fig. 1) and in situ hybridization (Figs. 5f, h). Although the majority of green receptors are double-peaked with a secondary peak in the UV (Fig. 2), we sometimes encountered single-peaked green receptors with very reduced UV sensitivity (Bandai et al. 1992). We later revealed that the single-peaked green receptors actually exist in the type II ommatidia (Arikawa et al. 1999a). The reduction of UV sensitivity can be explained by the pre-absorption of UV light by 3-hydroxyretinol at the distal portion of the type II ommatidia (Fig. 7c).
The combination of the various experimental approaches, namely anatomy, electrophysiology and molecular biology, combined with the modeling, has yielded the insight that the Papilio retina is composed of three different types of ommatidia, each containing a unique set of spectral receptors (Table 1). The basis of the spectral sensitivities of the photoreceptors is sometimes quite complicated, and often due to the combined effect of more than one visual pigment and the filtering by photostable pigments. The finally resulting spectral sensitivities have presumably been realized to optimize color vision. Discussions about the evolution of color vision, usually focus exclusively on the spectral tuning of visual pigment molecules based on their amino acid sequences (Chittka and Briscoe 2001; Gaertner 2000; Pichaud et al. 1999; Yokoyama and Yokoyama 2000). However, several other factors may play a role, as is demonstrated by the photoreceptors of Papilio. A similar organization as in Papilio, where multiple visual pigments are expressed in single cells or where spectral filtering sharpens the spectral sensitivities, may be encountered in many other animals, both in vertebrates (Lyubarsky et al. 1999; Makino and Dodd 1996) and in invertebrates (Marshall et al. 1991; Sakamoto et al. 1996). Flies utilize a different mechanism to broaden the spectral sensitivity of the peripheral (R1–6) photoreceptors. The photoreceptors contain a blue sensitive visual pigment (Rh1) with one molecule of 3-hydroxyretinol attached as a photostable sensitizing pigment. The sensitizing pigment absorbs UV light and transfers the energy to Rh1, resulting in another high sensitivity in the UV wavelength region in addition to the main sensitivity band peaking at around 480 nm (Stavenga et al. 2000).
The Papilio retina is a random mesh of three types of ommatidia, at least in the ventral half of the compound eye. Is this specific to Papilio, or is this arrangement shared by other insects? The phenomenon of ommatidial heterogeneity itself was repeatedly reported in flies (Franceschini et al. 1981; Hardie et al. 1981; Chou et al. 1996), a digger wasp (Ribi 1978), and in a moth (Meinecke and Langer 1984). Ommatidial heterogeneity was demonstrated in many butterfly species by their characteristic tapetal reflection (Bernard and Miller 1970; Stavenga et al. 2001). Stavenga (2002a) developed wide-angle telescope optics to study the ommatidial heterogeneity of butterflies into great detail, and thus clearly demonstrated the dorso-ventral specialization and species diversity of reflection colors (Stavenga 2002b). However, the eyes of papilionid butterflies form an exception by lacking the tapetum, and thus they do not exhibit the phenomenon of tapetal reflection.
The study of lepidopteran eyes has recently seen considerable progress. The eyes of the small white Pieris rapae (Pieridae) (Qiu et al. 2002; Qiu and Arikawa 2003a), the painted lady Vanessa cardui (Nymphalidae) (Briscoe et al. 2003), and the nocturnal tobacco hawkmoth Manduca sexta (Sphingidae) (White et al. 2003) all appear to contain at least three types of ommatidia in the ventral half. The striking similarity among these species is found in the combination of short-wavelength receptors in one of the three types of ommatidia. Typically, one type has two UV receptors, one has two blue receptors, and one has both UV and blue receptors (see Table 1 for the case of Papilio). Colocalization of UV and blue receptors in a single ommatidium suggests that these animals are able, in principle, to discriminate slight wavelength differences of a very small target in the short wavelength region of the spectrum. Actually, a day-active sphingid Macroglossum stellatrum can discriminate two similar spectral lights in the UV region (e.g., 365 nm and 380 nm) upon feeding (Kelber and Henique 1999). This property is probably important for nectar-feeding insects. In fact, our preliminary data have shown that the same photoreceptor organization occurs in the honeybee Apis mellifera (K. Arikawa, unpublished observations).
Of course, species differences certainly exist. If we define red receptors as the receptors with a rather sharp sensitivity profile peaking in the wavelength region longer than ca. 600 nm, the R5–8 of type I ommatidia of Papilio qualify as a type of red receptor (λmax=600 nm, Fig. 2), but bees do not possess such a type of receptor (Menzel and Backhaus 1989). Do all butterflies have some type of red receptor? The answer must be no, for at least two reasons. Firstly, we have shown by electrophysiology that two species of nymphalid butterflies, Polygonia c-aureum and Sasakia charonda, do not have photoreceptors with spectral sensitivities peaking in the red; their L-receptors peak in the green to orange wavelength regions (Kinoshita et al. 1997). Recent microspectroscopical and molecular biological investigations on Vanessa revealed that their eyes express only three types of visual pigments, with probable absorption peaks at 360, 470, and 530 nm (Briscoe et al. 2003). The situation in the diurnal Vanessa is quite similar to that of the nocturnal Manduca (White et al. 2003). Secondly, there are species that have more than one type of red receptor: we have identified two distinct long-wavelength (red) receptors, LW620 and LW640, peaking at 620 nm and 640 nm, respectively, in the eye of the male small white, Pieris rapae, by intracellular electrophysiology and subsequent histology (Qiu and Arikawa 2003b). The Pieris eye consists of three types of ommatidia that are easily distinguished by their perirhabdomeral pigmentation (Qiu et al. 2002). The LW620 receptors exist in the proximal tier of type I and III ommatidia, whereas the LW640 receptors were found exclusively in the proximal tier of type II ommatidia. Type II ommatidia express a denser red pigmentation compared to that of type I and III ommatidia, which is a possible cause of the difference in the spectral sensitivities between the two types of red receptors. Whether the opsins expressed in these receptors are identical or not is still an open question.
Another notable difference, at least at the present stage, is that coexpression of multiple visual pigments in single photoreceptors is exclusively found in the Papilio eye. We clearly demonstrated in Papilio that the R5–8 in type II ommatidia contain two functional visual pigments with different absorption spectra, resulting in a very wide spectral sensitivity (Fig. 9) (Arikawa et al. 2003). Is this specific to Papilio, or is it common in other animals? This question must await further molecular biological analyses.
The biological significance of the ommatidial heterogeneity and the randomness is still enigmatic. Accumulated data have indicated that green receptors are present in all ommatidia forming a complete hexagonal lattice (e.g., R3 and R4 of Papilio, Table 1), whereas other receptors distribute rather randomly. The regular hexagonal array of green receptors of course favors good spatial resolution. The random system of several spectral receptor types (including green receptors of R5–8, see Table 1) may be related to color vision, perhaps being required to avoid chromatic aliasing. The retinal randomness is also shared by primates, which have sophisticated color vision systems: a random distribution of M- and L-cones has been demonstrated in talapoin monkeys (Mollon and Bowmaker 1992) and also in living humans (Roorda and Williams 1999). Is the randomness essential for recognizing color? Probably not, because some fish and mantis shrimp, which have very regular retinae, have true color vision (Marshall et al. 1996; Neumeyer 1992), although fundamental differences may exist between the color vision systems of aquatic and terrestrial dwellers. At any rate, the retina of butterflies provides an excellent subject for studying the functional significance of the receptor mosaic for color vision and for visual acuity in general.
I first want to express my thanks to the editor of this journal for encouraging me to write this review. Drs D.G. Stavenga and M. Kinoshita, and two anonymous referees provided critical comments on the manuscript. The work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) and by the PRESTO program of the Japan Science Technology Corporation (JST).
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