Opsin Locus Research Articles

The evolution of red color vision is linked to coordinated rhodopsin tuning in lycaenid butterflies

Color vision has evolved multiple times in both vertebrates and invertebrates and is largely determined by the number and variation in spectral sensitivities of distinct opsin subclasses. However, because of the difficulty of expressing long-wavelength (LW) invertebrate opsins in vitro, our understanding of the molecular basis of functional shifts in opsin spectral sensitivities has been biased toward research primarily in vertebrates. This has restricted our ability to address whether invertebrate Gq protein-coupled opsins function in a novel or convergent way compared to vertebrate Gt opsins. Here we develop a robust heterologous expression system to purify invertebrate rhodopsins, identify specific amino acid changes responsible for adaptive spectral tuning, and pinpoint how molecular variation in invertebrate opsins underlie wavelength sensitivity shifts that enhance visual perception. By combining functional and optophysiological approaches, we disentangle the relative contributions of lateral filtering pigments from red-shifted LW and blue short-wavelength opsins expressed in distinct photoreceptor cells of individual ommatidia. We use in situ hybridization to visualize six ommatidial classes in the compound eye of a lycaenid butterfly with a four-opsin visual system. We show experimentally that certain key tuning residues underlying green spectral shifts in blue opsin paralogs have evolved repeatedly among short-wavelength opsin lineages. Taken together, our results demonstrate the interplay between regulatory and adaptive evolution at multiple Gq opsin loci, as well as how coordinated spectral shifts in LW and blue opsins can act together to enhance insect spectral sensitivity at blue and red wavelengths for visual performance adaptation.

Spectral sensitivity of guppy visual pigments reconstituted in vitro to resolve association of opsins with cone cell types

The guppy (Poecilia reticulata) shows remarkable variation of photoreceptor cells in the retina, especially those sensitive to middle-to-long wavelengths of light. Microspectrophotometry (MSP) has revealed varying “green”, “green-yellow” and “yellow” cone cells among guppies in Trinidad and Venezuela (Cumana). In the guppy genome, there are four “long-wave” opsin loci (LWS-1, -2, -3 and -4). Two LWS-1 alleles have potentially differing spectral sensitivity (LWS-1/180Ser and LWS-1/180Ala). In addition, two “middle-wave” loci (RH2-1 and -2), two “short-wave” loci (SWS2-A and -B), and a single “ultraviolet” locus (SWS1) as well as a single “rhodopsin” locus (RH1) are present. However, the absorption spectra of these photopigments have not been measured directly and the association of cell types with these opsins remains speculative. In the present study, we reconstituted these opsin photopigments in vitro. The wavelengths of maximal absorbance (λmax) were 571nm (LWS-1/180Ser), 562nm (LWS-1/180Ala), 519nm (LWS-3), 516nm (LWS-2), 516nm (RH2-1), 476nm (RH2-2), 438nm (SWS2-A), 408nm (SWS2-B), 353nm (SWS1) and 503nm (RH1). The λmax of LWS-3 is much shorter than the value expected (560nm) from the “five-sites” rule. The two LWS-1 alleles could explain difference of the reported MSP λmax values for the yellow cone class between Trinidad and Cumana guppies. Absence of the short-wave-shifted LWS-3 and the green-yellow cone in the green swordtail supports the hypothesis that this cell class of the guppy co-expresses the LWS-1 and LWS-3. These results reveal the basis of variability in the guppy visual system and provide insight into the behavior and ecology of these tropical fishes.

Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs

Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations.

Highly polymorphic colour vision in a New World monkey with red facial skin, the bald uakari (Cacajao calvus).

Colour vision is highly variable in New World monkeys (NWMs). Evidence for the adaptive basis of colour vision in this group has largely centred on environmental features such as foraging benefits for differently coloured foods or predator detection, whereas selection on colour vision for sociosexual communication is an alternative hypothesis that has received little attention. The colour vision of uakaris (Cacajao) is of particular interest because these monkeys have the most dramatic red facial skin of any primate, as well as a unique fission/fusion social system and a specialist diet of seeds. Here, we investigate colour vision in a wild population of the bald uakari,C. calvus, by genotyping the X-linked opsin locus. We document the presence of a polymorphic colour vision system with an unprecedented number of functional alleles (six), including a novel allele with a predicted maximum spectral sensitivity of 555 nm. This supports the presence of strong balancing selection on different alleles at this locus. We consider different hypotheses to explain this selection. One possibility is that trichromacy functions in sexual selection, enabling females to choose high-quality males on the basis of red facial coloration. In support of this, there is some evidence that health affects facial coloration in uakaris, as well as a high prevalence of blood-borne parasitism in wild uakari populations. Alternatively, the low proportion of heterozygous female trichromats in the population may indicate selection on different dichromatic phenotypes, which might be related to cryptic food coloration. We have uncovered unexpected diversity in the last major lineage of NWMs to be assayed for colour vision, which will provide an interesting system to dissect adaptation of polymorphic trichromacy.

Can colour vision re-evolve? Variation in the X-linked opsin locus of cathemeral Azara's owl monkeys (Aotus azarae azarae).

BackgroundDo evolutionary specializations lead to evolutionary constraint? This appears plausible, particularly when specialization leads to loss of complex adaptations. In the owl monkey lineage, nocturnality clearly arose from a diurnal ancestor. This behavioural shift was accompanied by morphological changes in the eye and orbit and complete loss of colour vision via missense mutations in the gene encoding the short-wave sensitive visual pigment (SWS opsin). Interestingly, at least one subspecies of owl monkey, Azara’s owl monkey (Aotus azarae azarae), has regained activity in daylight. Given that all primate species that are active in daylight, including primarily diurnal species and species that are active during both day and night, have at least dichromatic colour vision, it seems reasonable to propose that dichromacy would be adaptive in A. a. azarae. With a disabled SWS opsin, the main avenue available for Azara’s owl monkeys to re-evolve colour vision is via a polymorphism in the intact X-linked opsin locus, which commonly occurs in other New World monkeys. To examine this possibility we assayed variation in the X-linked opsin of A. a. azarae, focusing on the three exons (3, 4 and 5) that control spectral sensitivity.ResultsWe found low opsin genetic variation on a population level, and no differences at the three main sites that lead to variation in spectral sensitivity in the opsins of other New World monkeys. Two rare alleles with single amino acid variants are segregating in the population, but previous functional studies indicate that these are unlikely to affect spectral sensitivity.ConclusionsGenetic constraint on the re-evolution of colour vision is likely operating in Azara’s owl monkey, which may affect the niche that this subspecies is able to occupy.

Genetic diversity of color phenotypes in the koi (<i>Cyprinus carpio</i> L.) as identified by molecular markers

The common carp (Cyprinus carpio) has a large variety of strains. The more popular are the koi (Japanese ornamental carp), which are still bred today to generate creative colors and patterns, giving rise to multiple phenotypes. Since koi are in great demand, there is a challenge to determine the genetics defining their quality. Two methods: 1) direct sequencing of five candidate gene regions, i.e., mitochondrial (cytochrome b, 12S gene and the D-loop) and nuclear (red sensitive opsin and Rag-1) loci, to detect single nucleotide polymorphisms (SNP)s and 2) random amplification of polymorphic DNA (RAPD), were used to differentiate among four koi strains (Kohaku, Sanke, Ghost and Ohgon) and the common carp. Novel SNPs, distinguishing between koi and the common carp, were revealed in cytochrome b, the D-loop and in the red sensitive opsin; one was a missense mutation in cytochrome b at position 15860, in which threonine in the common carp became alanine in all koi strains examined. The Kohaku strain was found to have two alleles in the mitochondrial fragments, forming two different haplotypes (subpo-pulations). These novel SNPs distinguished between koi strains and the common carp, and the RAPD method enabled further differentiation among the four koi strains.

Allelic Variation in Malawi Cichlid Opsins: A Tale of Two Genera

The role of sequence variation in the spectral tuning of color vision is well established in many systems. This includes the cichlids of Lake Victoria where sequence variation has been linked to environmental light gradients and speciation. The cichlids of Lake Malawi are a similar model for visual evolution, but the role of gene sequence variation in visual tuning between closely related species is unknown. This work describes such variation in multiple species of two rock-dwelling genera: Metriaclima and Labidochromis. Genomic DNA for seven cone opsin genes was sequenced and the structure of the opsin proteins was inferred. Retinal binding pocket polymorphisms were identified and compared to available data regarding spectral absorbance shifts. Sequence variation with known or potential effects on absorbance spectra were found in four genes: SWS1 (UV sensitive), SWS2B (violet sensitive), RH2Abeta (green sensitive), and LWS (red sensitive). Functional variation was distributed such that each genus had both a variable short-wavelength and long-wavelength sensitive opsin. This suggests spectral tuning is important at the margins of the cichlid visual spectrum. Further, there are two SWS1 opsin alleles that differ in sensitivity by 10 nm and are >2 MY divergent. One of these occurs in a haplotype block >1 kb. Potential haplotype blocks were found around the RH2 opsin loci. These data suggest that molecular diversification has resulted in functionally unique alleles and changes to the visual system. These data also suggest that opsin sequence variation tunes spectral sensitivities between closely related species and that the specific regions of spectral tuning are genus-specific.

A Fish Eye Out of Water: Ten Visual Opsins in the Four-Eyed Fish, Anableps anableps

The “four-eyed” fish Anableps anableps has numerous morphological adaptations that enable above and below-water vision. Here, as the first step in our efforts to identify molecular adaptations for aerial and aquatic vision in this species, we describe the A. anableps visual opsin repertoire. We used PCR, cloning, and sequencing to survey cDNA using unique primers designed to amplify eight sequences from five visual opsin gene subfamilies, SWS1, SWS2, RH1, RH2, and LWS. We also used Southern blotting to count opsin loci in genomic DNA digested with EcoR1 and BamH1. Phylogenetic analyses confirmed the identity of all opsin sequences and allowed us to map gene duplication and divergence events onto a tree of teleost fish. Each of the gene-specific primer sets produced an amplicon from cDNA, indicating that A. anableps possessed and expressed at least eight opsin genes. A second PCR-based survey of genomic and cDNA uncovered two additional LWS genes. Thus, A. anableps has at least ten visual opsins and all but one were expressed in the eyes of the single adult surveyed. Among these ten visual opsins, two have key site haplotypes not found in other fish. Of particular interest is the A. anableps-specific opsin in the LWS subfamily, S180γ, with a SHYAA five key site haplotype. Although A. anableps has a visual opsin gene repertoire similar to that found in other fishes in the suborder Cyprinodontoidei, the LWS opsin subfamily has two loci not found in close relatives, including one with a key site haplotype not found in any other fish species. A. anableps opsin sequence data will be used to design in situ probes allowing us to test the hypothesis that opsin gene expression differs in the distinct ventral and dorsal retinas found in this species.

A butterfly eye's view of birds

The striking color patterns of butterflies and birds have long interested biologists. But how these animals see color is less well understood. Opsins are the protein components of the visual pigments of the eye. Color vision has evolved in butterflies through opsin gene duplications, through positive selection at individual opsin loci, and by the use of filtering pigments. By contrast, birds have retained the same opsin complement present in early-jawed vertebrates, and their visual system has diversified primarily through tuning of the short-wavelength-sensitive photoreceptors, rather than by opsin duplication or the use of filtering elements. Butterflies and birds have evolved photoreceptors that might use some of the same amino acid sites for generating similar spectral phenotypes across approximately 540 million years of evolution, when rhabdomeric and ciliary-type opsins radiated during the early Cambrian period. Considering the similarities between the two taxa, it is surprising that the eyes of birds are not more diverse. Additional taxonomic sampling of birds may help clarify this mystery.

CHX10 Targets a Subset of Photoreceptor Genes

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.

Differential Expression of Duplicated Opsin Genes in Two EyeTypes of Ostracod Crustaceans

In the first molecular study of ostracod (Crustacea) vision, we present partial cDNA sequences of ostracod visual pigment genes (opsins). We found strong support for differential expression of opsins in ostracod median and compound eyes and suggest that photoreceptor specific expression may be a general phenomenon in organisms with multiple receptors. We infer that eye-specific expression predates the divergence of the two species examined, Skogsbergia lerneri and Vargula hilgendorfii, because eye-specific opsin orthologs are present in both species. We found multiple opsin loci in ostracods, estimating that at least eight are present in Skogsbergia lerneri. All opsins from both ostracod species examined are more closely related to each other than to any other known opsin sequences. Because we find no evidence for gene conversion or alternative splicing, we suggest the occurrence of many recent gene duplications. Why ostracods may have retained multiple recent opsin gene duplicates is unknown, but we discuss several possible hypotheses.

The effect of selection on a long wavelength-sensitive (LWS) opsin gene of Lake Victoria cichlid fishes.

In East African Lake Victoria >200 endemic species of haplochromine fishes have been described on the basis of morphological and behavioral differences. Yet molecular analysis has failed to reveal any species-specific differences among these fishes in either mitochondrial or nuclear genes. Although the genes could be shown to vary, the variations represent trans-species polymorphisms not yet assorted along species lines. Nevertheless, fixed genetic differences must exist between the species at loci responsible for the adaptive characters distinguishing the various forms from one another. Here we describe variation and fixation at the long wavelength-sensitive (LWS) opsin locus, which is selection-driven, adaptive, and if not species- then at least population-specific. Because color is one of the characters distinguishing species of haplochromine fishes and color perception plays an important part in food acquisition and mate choice, we suggest that the observed variation and fixation at the LWS opsin locus may have been involved in the process that has led to the spectacular species divergence of haplochromine fishes in Lake Victoria.

The molecular evolution of visual pigments of freshwater crayfishes (Decapoda: Cambaridae).

This study examines the diverse maximum wavelength absorption (lambdamax) found in crayfishes (Decapoda: Cambaridae and Parastacidae) and the associated genetic variation in their opsin locus. We measured the wavelength absorption in the photoreceptors of six species that inhabit environments of different light intensities (i.e., burrows, streams, standing waters, and subterranean waters). Our results indicate that there is relatively little variation in lambdamax (522-530 nm) among species from different genera and families. The existing variation did not correlate with the habitat differences of the crayfishes studied. We simultaneously sequenced the rhodopsin gene to identify the amino acid replacements that affect shifts in maximum wavelength absorption. We then related these to changes that correlated with shifts in lambdamax by reconstructing ancestral character states using a maximum-likelihood approach. Using amino acid sequences obtained from five species (all were 301 amino acids in length), we identified a number of candidates for producing shifts of 4 to 8 nm in lambdamax. These amino acid replacements occurred in similar regions to those involved in spectral shifts in vertebrates.

A New Form of Inherited Red-Blindness Identified in Zebrafish

A red-blind zebrafish mutant, partial optokinetic response b (pob), has been isolated by measuring eye movements of larvae in a three-generation screen for recessive mutations affecting the visual system. pob larvae exhibit eye movements in response to rotating black and white stripes illuminated with white light, but they do not move their eyes when the stripes are illuminated with red light. Physiological, immunohistochemical, and in situ hybridization analyses of pob retinas showed a selective loss of red-sensitive cones at 5 days postfertilization (dpf). At 3 dpf, cells expressing red opsin are present, suggesting that red-sensitive cones form initially but then disappear rapidly, whereas other photoreceptors remain. Linkage analysis indicated that the mutation identified in the pob mutant is not at the red opsin locus. Because red opsin is the only known molecule unique to red cones, these data suggest that a novel gene is required for the maintenance or function of red cones.

RER, an Evolutionarily Conserved Sequence Upstream of the Rhodopsin Gene, Has Enhancer Activity

Previous transgenic mouse experiments localized the mammalian rhodopsin gene promoter to a region just upstream of the mRNA start site, and also suggested the existence of a second more distal regulatory region. A highly conserved 100-base pair (bp) sequence which is homologous to the red and green opsin locus control region is located 1.5-2 kilobases upstream of the rhodopsin gene (depending on the species). In order to test the activity of this 100-bp region, transgenic mice were generated with bovine rhodopsin promoter/lacZ constructs which differed only by the presence or absence of the sequence. Of 11 lines generated, all demonstrated photoreceptor-specific expression of the transgene, but the lines with the putative regulatory region showed significantly higher expression. Additional transgenic lines in which the region was fused to a minimal heterologous promoter did not show transgene expression in the retina. Gel mobility shift and DNase I footprint assays demonstrated that bovine retinal nuclear extracts contain retina-specific as well as ubiquitously expressed factors that interact with the putative regulatory region in a sequence-specific manner. These results indicate that the 100-bp sequence can indeed function in vivo as a rhodopsin enhancer region.

Why ontogeny recapitulates phylogeny.

In development, mammalian embryos successively develop notochord, somites, gill pouches and three sets of kidneys: pronephros, mesonephros and metanephros. Hence, Ernst Haeckel's dictum "ontogeny recapitulates phylogeny." The reason for this dictum appears to be the immortality of genes. Genes, once well entrenched, appear to linger for a long time even after the forfeiture of their raison d'être. The genes for dental enamel and dentin of the chicken are good examples. Furthermore, genes once doomed may resurrect themselves. For example, genes for red and green opsins of higher primates arose as alleles of the once doomed but resurrected red opsin locus.