Immunohistochemical localization of Papilio RBP in the eye of butterflies

We recently identified a novel retinoid binding protein, Papilio RBP, in the soluble fraction of the eye homogenate of the butterfly Papilio xuthus, and demonstrated that the protein is involved in the visual cycle. We now have localized the protein in the Papilio eye by light and electron microscopic immunohistochemistry using a monospecific antiserum produced against artificially expressed Papilio RBP. We found strong immunoreactivity in the primary as well as secondary pigment cells and in the tracheal cells. The pigment cells have long been regarded as an important site of the visual cycle, and this view is further supported by the present result. Interestingly, the cytoplasm and nuclei of these cells were equally labeled, indicating that the protein exists in both the cytoplasm and the nucleus. We conducted a survey for the existence of the Papilio RBP-like proteins in other insects including several species of butterflies, dragonflies, cicadas, grasshoppers and honeybees. Anti-Papilio RBP immunoreactivity was confirmed in the proteins isolated only from butterflies belonging to the superfamily Papilionoidea and not from other species. In all insects tested, however, fluorescing proteins were clearly detected, suggesting that these insects also have similar retinol-binding proteins

A unique visual pigment expressed in green, red and deep-red receptors in the eye of the small white butterfly, Pieris rapae crucivora

The full primary structure of a long-wavelength absorbing visual pigment of the small white butterfly, Pieris rapae crucivora, was determined by molecular cloning. In situ hybridization of the opsin mRNA of the novel visual pigment (PrL) demonstrated that it is expressed in the two distal photoreceptor cells (R3 and R4) as well as in the proximal photoreceptors (R5–8) in all three types of ommatidia of the Pieris eye. The main, long-wavelength band of the spectral sensitivities of the R3 and R4 photoreceptors is well described by the absorption spectrum of a visual pigment with absorption maximum at 563 nm; i.e. PrL is a visual pigment R563. The spectral sensitivities of R5–8 photoreceptors in ommatidial type I and III peak at 620 nm and those in type II ommatidia peak at 640 nm. The large shifts of the spectral sensitivities of the R5–8 photoreceptors with respect to the absorption spectrum of their visual pigment can be explained with the spectral filtering by pale-red (PR) and deep-red (DR) screening pigments that are concentrated in clusters of granules near the rhabdom boundary. The peak absorbance of the two spectral filters appears to be approximately 1 (PR) and 2 (DR)

Eyes with basic dorsal and specific ventral regions in the glacial Apollo, Parnassius glacialis (Papilionidae)

Recent studies on butterflies have indicated that their colour vision system is almost species specific. To address the question of how this remarkable diversity evolved, we investigated the eyes of the glacial Apollo, Parnassius glacialis, a living fossil species belonging to the family Papilionidae. We identified four opsins in the Parnassius eyes – an ultraviolet- (PgUV), a blue- (PgB), and two long wavelength (PgL2, PgL3)-absorbing types – and localized their mRNAs within the retina. We thus found ommatidial heterogeneity and a clear dorso-ventral regionalization of the eye. The dorsal region consists of three basic types of ommatidia that are similar to those found in other insects, indicating that this dorsal region retains the ancestral state. In the ventral region, we identified two novel phenomena: co-expression of the opsins of the UV- and B-absorbing type in a subset of photoreceptors, and subfunctionalization of long-wavelength receptors in the distal tier as a result of differential expression of the PgL2 and PgL3 mRNAs. Interestingly, butterflies from the closely related genus Papilio (Papilionidae) have at least three long-wavelength opsins, L1–L3. The present study indicates that the duplication of L2 and L3 occurred before the Papilio lineage diverged from the rest, whereas L1 was produced from L3 in the Papilio lineage

Opsin expression, physiological characterization and identification of photoreceptor cells in the dorsal rim area and main retina of the desert locust, Schistocerca gregaria.

For compass orientation many insects rely on the pattern of sky polarization, but some species also exploit the sky chromatic contrast. Desert locusts, Schistocerca gregaria, detect polarized light through a specialized dorsal rim area (DRA) in their compound eye. To better understand retinal mechanisms underlying visual navigation, we compared opsin expression, spectral and polarization sensitivities and response-stimulus intensity functions in the DRA and main retina of the locust. In addition to previously characterized opsins of long-wavelength-absorbing (Lo1) and blue-absorbing visual pigments (Lo2), we identified an opsin of an ultraviolet-absorbing visual pigment (LoUV). DRA photoreceptors exclusively expressed Lo2, had peak spectral sensitivities at 441 nm and showed high polarization sensitivity (PS 1.3-31.7). In contrast, ommatidia in the main eye co-expressed Lo1 and Lo2 in five photoreceptors, expressed Lo1 in two proximal photoreceptors, and Lo2 or LoUV in one distal photoreceptor. Correspondingly, we found broadband blue- and green-peaking spectral sensitivities in the main eye and one narrowly tuned UV peaking receptor. Polarization sensitivity in the main retina was low (PS 1.3-3.8). V-log I functions in the DRA were steeper than in the main retina, supporting a role in polarization vision. Desert locusts occur as two morphs, a day-active gregarious and a night-active solitarious form. In solitarious locusts, sensitivities in the main retina were generally shifted to longer wavelengths, particularly in ventral eye regions, supporting a nocturnal lifestyle at low light levels. The data support the role of the DRA in polarization vision and suggest trichromatic colour vision in the desert locust

Evolution and mechanism of spectral tuning of Blue-absorbing visual pigments in butterflies

The eyes of flower-visiting butterflies are often spectrally highly complex with multiple opsin genes generated by gene duplication, providing an interesting system for a comparative study of color vision. The Small White butterfly, Pieris rapae, has duplicated blue opsins, PrB and PrV, which are expressed in the blue (λmax = 453 nm) and violet receptors (λmax = 425 nm), respectively. To reveal accurate absorption profiles and the molecular basis of the spectral tuning of these visual pigments, we successfully modified our honeybee opsin expression system based on HEK293s cells, and expressed PrB and PrV, the first lepidopteran opsins ever expressed in cultured cells. We reconstituted the expressed visual pigments in vitro, and analysed them spectroscopically. Both reconstituted visual pigments had two photointerconvertible states, rhodopsin and metarhodopsin, with absorption peak wavelengths 450 nm and 485 nm for PrB and 420 nm and 482 nm for PrV. We furthermore introduced site-directed mutations to the opsins and found that two amino acid substitutions, at positions 116 and 177, were crucial for the spectral tuning. This tuning mechanism appears to be specific for invertebrates and is partially shared by other pierid and lycaenid butterfly species

Spectral Organization of Ommatidia in Flower-visiting Insects

This article reviews recent advances of studies on the spectral organization of the compound eye in lepidopteran and hymenopteran insects. The compound eyes consist of ommatidia, which contain a set of photoreceptor cells. The common feature is that the ommatidia can be divided into three types, according to the combination of spectral classes of photoreceptors. Honeybees and nymphalid butterflies provide the simplest case with three photoreceptor classes having peak sensitivities in the ultraviolet (UV), blue (B), or green (G) wavelength region. These receptors populate the ommatidia in fixed combinations. In type I ommatidia, the main eight photoreceptors consist of one UV, one B, and six G receptors. Type II has two UV and six G receptors, and type III has two B and six G receptors. The organization is basically retained in all insect species studied so far, although some butterflies have more than six classes of spectral receptors, including those sensitive in the violet and red wavelength regions. To acquire these additional receptors, two distinct strategies are applied: the multiple opsin strategy, taken by the Japanese yellow swallowtail butterfly Papilio xuthus, and the filter strategy, used by the small white butterfly Pieris rapae.

Unique Temporal Expression of Triplicated Long-Wavelength Opsins in Developing Butterfly Eyes

Following gene duplication events, the expression patterns of the resulting gene copies can often diverge both spatially and temporally. Here we report on gene duplicates that are expressed in distinct but overlapping patterns, and which exhibit temporally divergent expression. Butterflies have sophisticated color vision and spectrally complex eyes, typically with three types of heterogeneous ommatidia. The eyes of the butterfly Papilio xuthus express two green- and one red-absorbing visual pigment, which came about via gene duplication events, in addition to one ultraviolet (UV)- and one blue-absorbing visual pigment. We localized mRNAs encoding opsins of these visual pigments in developing eye disks throughout the pupal stage. The mRNAs of the UV and blue opsin are expressed early in pupal development (pd), specifying the type of the ommatidium in which they appear. Red sensitive photoreceptors first express a green opsin mRNA, which is replaced later by the red opsin mRNA. Broadband photoreceptors (that coexpress the green and red opsins) first express the green opsin mRNA, later change to red opsin mRNA and finally re-express the green opsin mRNA in addition to the red mRNA. Such a unique temporal and spatial expression pattern of opsin mRNAs may reflect the evolution of visual pigments and provide clues toward understanding how the spectrally complex eyes of butterflies evolved

A unique visual pigment expressed in green, red and deep-red receptors in the eye of the small white butterfly, Pieris rapae crucivora

The full primary structure of a long-wavelength absorbing visual pigment of the small white butterfly, Pieris rapae crucivora, was determined by molecular cloning. In situ hybridization of the opsin mRNA of the novel visual pigment (PrL) demonstrated that it is expressed in the two distal photoreceptor cells (R3 and R4) as well as in the proximal photoreceptors (R5–8) in all three types of ommatidia of the Pieris eye. The main, long-wavelength band of the spectral sensitivities of the R3 and R4 photoreceptors is well described by the absorption spectrum of a visual pigment with absorption maximum at 563 nm; i.e. PrL is a visual pigment R563. The spectral sensitivities of R5–8 photoreceptors in ommatidial type I and III peak at 620 nm and those in type II ommatidia peak at 640 nm. The large shifts of the spectral sensitivities of the R5–8 photoreceptors with respect to the absorption spectrum of their visual pigment can be explained with the spectral filtering by pale-red (PR) and deep-red (DR) screening pigments that are concentrated in clusters of granules near the rhabdom boundary. The peak absorbance of the two spectral filters appears to be approximately 1 (PR) and 2 (DR).