Reconstructing the ancestral butterfly eye: focus on the opsins.

The eyes of butterflies are remarkable, because they are nearly as diverse as the colors of wings. Much of eye diversity can be traced to alterations in the number, spectral properties and spatial distribution of the visual pigments. Visual pigments are light-sensitive molecules composed of an opsin protein and a chromophore. Most butterflies have eyes that contain visual pigments with a wavelength of peak absorbance, lambda(max), in the ultraviolet (UV, 300-400 nm), blue (B, 400-500 nm) and long wavelength (LW, 500-600 nm) part of the visible light spectrum, respectively, encoded by distinct UV, B and LW opsin genes. In the compound eye of butterflies, each individual ommatidium is composed of nine photoreceptor cells (R1-9) that generally express only one opsin mRNA per cell, although in some butterfly eyes there are ommatidial subtypes in which two opsins are co-expressed in the same photoreceptor cell. Based on a phylogenetic analysis of opsin cDNAs from the five butterfly families, Papilionidae, Pieridae, Nymphalidae, Lycaenidae and Riodinidae, and comparative analysis of opsin gene expression patterns from four of the five families, I propose a model for the patterning of the ancestral butterfly eye that is most closely aligned with the nymphalid eye. The R1 and R2 cells of the main retina expressed UV-UV-, UV-B- or B-B-absorbing visual pigments while the R3-9 cells expressed a LW-absorbing visual pigment. Visual systems of existing butterflies then underwent an adaptive expansion based on lineage-specific B and LW opsin gene multiplications and on alterations in the spatial expression of opsins within the eye. Understanding the molecular sophistication of butterfly eye complexity is a challenge that, if met, has broad biological implications.

Briscoe AD 《JOURNAL OF EXPERIMENTAL BIOLOGY》

Beauty in the eye of the beholder: the two blue opsins of lycaenid butterflies and the opsin gene-driven evolution of sexually dimorphic eyes.

Although previous investigations have shown that wing coloration is an important component of social signaling in butterflies, the contribution of opsin evolution to sexual wing color dichromatism and interspecific divergence remains largely unexplored. Here we report that the butterfly Lycaena rubidus has evolved sexually dimorphic eyes due to changes in the regulation of opsin expression patterns to match the contrasting life histories of males and females. The L. rubidus eye contains four visual pigments with peak sensitivities in the ultraviolet (UV; lambdamax=360 nm), blue (B; lambdamax=437 nm and 500 nm, respectively) and long (LW; lambdamax=568 nm) wavelength range. By combining in situ hybridization of cloned opsin-encoding cDNAs with epi-microspectrophotometry, we found that all four opsin mRNAs and visual pigments are expressed in the eyes in a sex-specific manner. The male dorsal eye, which contains only UV and B (lambdamax=437 nm) visual pigments, indeed expresses two short wavelength opsin mRNAs, UVRh and BRh1. The female dorsal eye, which also has the UV and B (lambdamax=437 nm) visual pigments, also contains the LW visual pigment, and likewise expresses UVRh, BRh1 and LWRh mRNAs. Unexpectedly, in the female dorsal eye, we also found BRh1 co-expressed with LWRh in the R3-8 photoreceptor cells. The ventral eye of both sexes, on the other hand, contains all four visual pigments and expresses all four opsin mRNAs in a non-overlapping fashion. Surprisingly, we found that the 500 nm visual pigment is encoded by a duplicate blue opsin gene, BRh2. Further, using molecular phylogenetic methods we trace this novel blue opsin gene to a duplication event at the base of the Polyommatine+Thecline+Lycaenine radiation. The blue opsin gene duplication may help explain the blueness of blue lycaenid butterflies.

Sison-Mangus MP ，Bernard GD ，Lampel J ，Briscoe AD ... -  《JOURNAL OF EXPERIMENTAL BIOLOGY》

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 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.

Arikawa K ，Iwanaga T ，Wakakuwa M ，Kinoshita M ... -  《Frontiers in Neural Circuits》

Adult stemmata of the butterfly Vanessa cardui express UV and green opsin mRNAs.

Adult stemmata are distinctive insect photoreceptors located on the posterior surfaces of the optic lobes. They originate as larval eyes that migrate inward during metamorphosis. We used a combination of light microscopy and in situ hybridization to examine their anatomical organization in the butterfly Vanessa cardui and to test for the presence of visual pigments, the light sensitive components of the visual transduction pathway. The bilateral cluster of six internal stemmata is located near the ventral edge of the lamina. They retain the dark screening pigment and overlying crystalline cones of the larval stemmata. We found two opsin mRNAs expressed in the stemmata that are also expressed, respectively, in UV-sensitive and green-sensitive photoreceptor cells in the compound eye. A third mRNA that is expressed in blue-sensitive photoreceptor cells of the compound eye was not expressed in the stemmata. Our results reinforce the idea that the adult stemmata are not merely developmental remnants of larval eyes, but remain functional, possibly as components of the circadian input channel.

Briscoe AD ，White RH 《CELL AND TISSUE RESEARCH》

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

Spectral tuning of rhodopsins commonly refers to the effects of opsin amino acid substitutions on the wavelength for peak sensitivity of the rhodopsin absorption spectrum. Nymphalini butterflies provide an opportunity for identifying some of the amino acid substitutions responsible for insect rhodopsin spectral tuning because the majority of photoreceptor cells (R3-9) in the adult retina express only a single long wavelength-sensitive (LWS) opsin mRNA transcript. Therefore, the opsin genotype can be directly correlated with its phenotype. We determined the LWS opsin gene sequence from cDNA of the mourning cloak Nymphalis antiopa, and from genomic DNA of the malachite Siproeta stelenes and the peacock Inachis io. Using an epi-microspectrophotometer we examined each butterfly's eyeshine for photochemical evidence of multiple LWS rhodopsins and found only one. We then performed partial-bleaching experiments to obtain absorbance spectra for the LWS rhodopsins of all three species as well as from another nymphalid, the buckeye Junonia coenia. The isolated LWS opsin gene sequences varied in length from 1437-1612 bp and encode rhodopsins R522 (S. stelenes), R530 (I. io), R534 (N. antiopa) and, together with a previously published sequence, R510 (J. coenia). Comparative sequence analysis indicates that the S. stelenes rhodopsin is slightly blue-shifted compared to the typical 530 nm lepidopteran rhodopsin because of the presence of a S138A substitution at a homologous site that in mammalian MWS/LWS rhodopsins causes a 5 nm blue-shift. The difference in peak absorption between R522 of S. stelenes and R530 of Inachis io is therefore largely accounted for by this substitution. This suggests that spectral tuning mechanisms employing the S138A may have evolved in parallel in mammalian and butterfly MWS/LWS rhodopsins across 500 million years of evolution.

Briscoe AD ，Bernard GD 《JOURNAL OF EXPERIMENTAL BIOLOGY》