Butterfly eyes are furnished with a variety of photoreceptors of different spectral sensitivities often in species-specific manner. We have conducted an extensive comparative study to address the question of how their spectrally complex retinas evolved. Here we investigated the structure and function of the eye of the common bluebottle butterfly (Graphium sarpedon), using electrophysiological, anatomical, and molecular approaches. Intracellular electrophysiology revealed that the eye contains photoreceptors of 15 distinct spectral sensitivities. These can be divided into six spectral receptor classes: ultraviolet-(UV), violet-(V), blue-(B), blue-green-(BG), green-(G), and red-(R) sensitive. The B, G, and R classes respectively contain three, four, and five subclasses. Fifteen is the record number of spectral receptors so far reported in a single insect eye. We localized these receptors by injecting dye into individual photoreceptors after recording their spectral sensitivities. We thus found that four of them are confined to the dorsal region, eight to the ventral, and three exist throughout the eye; the ventral eye region is spectrally richer than the dorsal region. We also identified mRNAs encoding visual pigment opsins of one ultraviolet, one blue, and three long wavelength-absorbing types. Localization of these mRNAs by in situ hybridization revealed that the dorsal photoreceptors each express a single opsin mRNA, but more than half of the ventral photoreceptors coexpress two or three L opsin mRNAs. This expression pattern well explains the spectral organization of the Graphium compound eye.

This paper documents the molecular organization of the eye of the Eastern Pale Clouded Yellow butterfly, Colias erate (Pieridae). We cloned four cDNAs encoding visual pigment opsins, corresponding to one ultraviolet, two blue and one long wavelength-absorbing visual pigments. Duplication of the blue visual pigment class occurs also in another pierid species, Pieris rapae, suggesting that blue duplication is a general feature in the family Pieridae. We localized the opsin mRNAs in the Colias retina by in situ hybridization. Among the nine photoreceptor cells in an ommatidium, R1-9, we found that R3-8 expressed the long wavelength class mRNA in all ommatidia. R1 and R2 expressed mRNAs of the short wavelength opsins in three fixed combinations, corresponding to three types of ommatidia. While the duplicated blue opsins in Pieris are separately expressed in two subsets of R1-2 photoreceptors, one blue sensitive and another violet sensitive, those of Colias appear to be always coexpressed.

Stomatopod crustaceans possess some of the most complex animal visual systems, including at least 16 spectrally distinct types of photoreceptive units (e.g., assemblages of photoreceptor cells). Here we fully characterize the set of opsin genes expressed in retinal tissues and determine expression patterns of each in the stomatopod Neogonodactylus oerstedii. Using a combination of transcriptome and RACE sequencing, we identified 33 opsin transcripts expressed in each N. oerstedii eye, which are predicted to form 20 long-wavelength–sensitive, 10 middle-wavelength–sensitive, and three UV-sensitive visual pigments. Observed expression patterns of these 33 transcripts were highly unusual in five respects: 1) All long-wavelength and short/middle-wavelength photoreceptive units expressed multiple opsins, while UV photoreceptor cells expressed single opsins; 2) most of the long-wavelength photoreceptive units expressed at least one middle-wavelength–sensitive opsin transcript; 3) the photoreceptors involved in spatial, motion, and polarization vision expressed more transcripts than those involved in color vision; 4) there is a unique opsin transcript that is expressed in all eight of the photoreceptive units devoted to color vision; and 5) expression patterns in the peripheral hemispheres of the eyes suggest visual specializations not previously recognized in stomatopods. Elucidating the expression patterns of all opsin transcripts expressed in the N. oerstedii retina reveals the potential for previously undocumented functional diversity in the already complex stomatopod eye and is a first step toward understanding the functional significance of the unusual abundance of opsins found in many arthropod species’ visual systems.

The tiered ommatidia of the Eastern Pale Clouded yellow butterfly, Colias erate, contain nine photoreceptor cells, four of which contribute their rhabdomeral microvilli to the distal tier of the rhabdom. We analyzed the visual pigments and spectral sensitivities of these distal photoreceptors in both sexes of Colias erate. A subset of photoreceptor cells expresses a newly discovered middle wavelength-absorbing opsin, Colias erate Blue (CeB), in addition to two previously described middle wavelength-absorbing opsins, CeV1 and CeV2. The other photoreceptors either coexpress CeV1 and CeV2, or exclusively express a short wavelength-absorbing opsin, CeUV, or a long wavelength-absorbing opsin, CeL. Males and females have the same visual pigment expression patterns, but the photoreceptor spectral sensitivities are sexually dimorphic. The photoreceptors coexpressing three middle wavelength-absorbing opsins are broad-blue receptors in males, but in females they are narrow-blue receptors. Those with CeV1 and CeV2 are violet receptors in females, while they are shouldered-blue receptors in males. The sexual dimorphism in spectral sensitivity is caused by a sex-specific distribution of fluorescent pigment that functions as a spectral filter.

SUMMARYRecent 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.