Estimated absorbance spectra of the visual pigments of the North Atlantic right whale (Eubalaena glacialis)

To assess the spectral sensitivities of the retinal visual pigments from the North Atlantic right whale (Eubalaena glacialis), we have cloned and sequenced two exons from the rod opsin gene and two exons from the middle‐wavelength sensitive (MWS) cone opsin gene in order to determine the amino acids at positions known to be key regulators of the spectral location of the absorbance maximum (λ_max). Based on previous mutagenesis models we estimate that the right whale possesses a rod visual pigment with a λ_max of 499 nm and a MWS cone visual pigment with a λ_max of 524 nm. Although the MWS cone visual pigment from the right whale is blue‐shifted in its spectral sensitivity like those from odontocetes, the spectral sensitivity of the right whale rod visual pigment is similar to those from terrestrial mammals.     

Switch in rod opsin gene expression in the European eel,Anguilla anguilla (L.).

The rod photoreceptors of the European eel, Anguilla anguilla (L.), alter their wavelength of maximum sensitivity (lambda max) from c.a. 523 nm to c.a. 482 nm at maturation, a switch involving the synthesis of a new visual pigment protein (opsin) that is inserted into the outer segments of existing rods. We artificially induced the switch in rod opsin production by the administration of hormones, and monitored the switch at the level of mRNA accumulation using radiolabelled oligonuleotides that hybridized differently to the two forms of eel rod opsin. The production of the deep-sea form of rod opsin was detected 6 h after the first hormone injection, and the switch in rod opsin expression was complete within four weeks, at which time only the mRNA for the deep-sea opsin was detectable in the retinal cells. It is suggested that this system could be used as a tractable model for studying the regulatory control of opsin gene expression.     

Visual responses in mice lacking critical components of all known retinal phototransduction cascades

The mammalian visual system relies upon light detection by outer-retinal rod/cone photoreceptors and melanopsin-expressing retinal ganglion cells. Gnat1(-/-);Cnga3(-/-);Opn4(-/-) mice lack critical elements of each of these photoreceptive mechanisms via targeted disruption of genes encoding rod α transducin (Gnat1); the cone-specific α3 cyclic nucleotide gated channel subunit (Cnga3); and melanopsin (Opn4). Although assumed blind, we show here that these mice retain sufficiently widespread retinal photoreception to drive a reproducible flash electroretinogram (ERG). The threshold sensitivity of this ERG is similar to that of cone-based responses, however it is lost under light adapted conditions. Its spectral efficiency is consistent with that of rod opsin, but not cone opsins or melanopsin, indicating that it originates with light absorption by the rod pigment. The TKO light response survives intravitreal injection of U73122 (a phospholipase C antagonist), but is inhibited by a missense mutation of cone α transducin (Gnat2(cpfl3)), suggesting Gnat2-dependence. Visual responses in TKO mice extend beyond the retina to encompass the lateral margins of the lateral geniculate nucleus and components of the visual cortex. Our data thus suggest that a Gnat1-independent phototransduction mechanism downstream of rod opsin can support relatively widespread responses in the mammalian visual system. This anomalous rod opsin-based vision should be considered in experiments relying upon Gnat1 knockout to silence rod phototransduction.     

Mechanisms of wavelength tuning in the rod opsins of deep-sea fishes.

The main object of this study was to investigate the molecular basis for changes in the spectral sensitivity of the visual pigments of deep-sea fishes. The four teleost species studied, Hoplostethus mediterraneus, Cataetyx laticeps, Gonostoma elongatum and Histiobranchus bathybius, are phylogenetically distant from each other and live at depths ranging from 500 to almost 5000 m. A single fragment of the intronless rod opsin gene was PCR-amplified from each fish and sequenced. The wavelength of peak sensitivity for the rod visual pigments of the four deep-sea species varies from 483 nm in H. mediterraneus and G. elongatum to 468 nm in C. laticeps. Six amino acids at sites on the inner face of the chromophore-binding pocket formed by the seven transmembrane a-helices are identified as candidates for spectral tuning. Substitutions at these sites involve either a change of charge, or a gain or loss of a hydroxyl group. Two of these, at positions 83 and 292, are consistently substituted in the visual pigments of all four species and are likely to be responsible for the shortwave sensitivity of the pigments. Shifts to wavelengths shorter than 480 nm may involve substitution at one or more of the remaining four sites. None of the modifications found in the derived sequences of these opsins suggest functional adaptations, such as increased content of hydroxyl-bearing or proline residues, to resist denaturation by the elevated hydrostatic pressures of the deep sea. Phylogenetic evidence for the duplication of the rod opsin gene in the Anguilliform lineage is presented.     

Cone opsin genes of african cichlid fishes: tuning spectral sensitivity by differential gene expression

Spectral tuning of visual pigments is typically accomplished through changes in opsin amino acid sequence. Within a given opsin class, changes at a few key sites control wavelength specificity. To investigate known differences in the visual pigment spectral sensitivity of the Lake Malawi cichlids, Metriaclima zebra (368, 488, and 533 nm) and Dimidiochromis compressiceps (447, 536, and 569 nm), we sequenced cone opsin genes from these species as well as Labeotropheus fuelleborni and Oreochromis niloticus. These cichlids have five distinct classes of cone opsin genes, including two unique SWS-2 genes. Comparisons of the inferred amino acid sequences from the five cone opsin genes of M. zebra, D. compressiceps, and L. fuelleborni show the sequences to be nearly identical. Therefore, evolution of key opsin sites cannot explain the differences in visual pigment sensitivities. Real-time PCR demonstrates that different cichlid species express different subsets of the available cone opsin genes. Metriaclima zebra and L. fuelleborni express a complement of genes which give them UV-shifted visual pigments, while D. compressiceps expresses a different set to produce a red-shifted visual system. Thus, variations in cichlid spectral sensitivity have arisen through evolution of gene regulation, rather than through changes in opsin amino acid sequence.     

Visual pigment evolution in Characiformes: The dynamic interplay of teleost whole‐genome duplication,surviving opsins and spectral tuning

Vision represents an excellent model for studying adaptation, given the genotype‐to‐phenotype map that has been characterized in a number of taxa. Fish possess a diverse range of visual sensitivities and adaptations to underwater light, making them an excellent group to study visual system evolution. In particular, some speciose but understudied lineages can provide a unique opportunity to better understand aspects of visual system evolution such as opsin gene duplication and neofunctionalization. In this study, we showcase the visual system evolution of neotropical Characiformes and the spectral tuning mechanisms they exhibit to modulate their visual sensitivities. Such mechanisms include gene duplications and losses, gene conversion, opsin amino acid sequence and expression variation, and A₁/A₂‐chromophore shifts. The Characiforms we studied utilize three cone opsin classes (SWS2, RH2, LWS) and a rod opsin (RH1). However, the characiform's entire opsin gene repertoire is a product of dynamic evolution by opsin gene loss (SWS1, RH2) and duplication (LWS, RH1). The LWS‐ and RH1‐duplicates originated from a teleost specific whole‐genome duplication as well as characiform‐specific duplication events. Both LWS‐opsins exhibit gene conversion and, through substitutions in key tuning sites, one of the LWS‐paralogues has acquired spectral sensitivity to green light. These sequence changes suggest reversion and parallel evolution of key tuning sites. Furthermore, characiforms' colour vision is based on the expression of both LWS‐paralogues and SWS2. Finally, we found interspecific and intraspecific variation in A₁/A₂‐chromophores proportions, correlating with the light environment. These multiple mechanisms may be a result of the diverse visual environments where Characiformes have evolved.     

The Giant Mottled Eel,Anguilla marmorata,Uses Blue-Shifted Rod Photoreceptors during Upstream Migration

Catadromous fishes migrate between ocean and freshwater during particular phases of their life cycle. The dramatic environmental changes shape their physiological features, e.g. visual sensitivity, olfactory ability, and salinity tolerance. Anguilla marmorata, a catadromous eel, migrates upstream on dark nights, following the lunar cycle. Such behavior may be correlated with ontogenetic changes in sensory systems. Therefore, this study was designed to identify changes in spectral sensitivity and opsin gene expression of A. marmorata during upstream migration. Microspectrophotometry analysis revealed that the tropical eel possesses a duplex retina with rod and cone photoreceptors. The λ_max of rod cells are 493, 489, and 489 nm in glass, yellow, and wild eels, while those of cone cells are 508, and 517 nm in yellow, and wild eels, respectively. Unlike European and American eels, Asian eels exhibited a blue-shifted pattern of rod photoreceptors during upstream migration. Quantitative gene expression analyses of four cloned opsin genes (Rh1f, Rh1d, Rh2, and SWS2) revealed that Rh1f expression is dominant at all three stages, while Rh1d is expressed only in older yellow eel. Furthermore, sequence comparison and protein modeling studies implied that a blue shift in Rh1d opsin may be induced by two known (N83, S292) and four putative (S124, V189, V286, I290) tuning sites adjacent to the retinal binding sites. Finally, expression of blue-shifted Rh1d opsin resulted in a spectral shift in rod photoreceptors. Our observations indicate that the giant mottled eel is color-blind, and its blue-shifted scotopic vision may influence its upstream migration behavior and habitat choice.     

Spectral tuning by opsin coexpression in retinal regions that view different parts of the visual field

Vision frequently mediates critical behaviours, and photoreceptors must respond to the light available to accomplish these tasks. Most photoreceptors are thought to contain a single visual pigment, an opsin protein bound to a chromophore, which together determine spectral sensitivity. Mechanisms of spectral tuning include altering the opsin, changing the chromophore and incorporating pre-receptor filtering. A few exceptions to the use of a single visual pigment have been documented in which a single mature photoreceptor coexpresses opsins that form spectrally distinct visual pigments, and in these exceptions the functional significance of coexpression is unclear. Here we document for the first time photoreceptors coexpressing spectrally distinct opsin genes in a manner that tunes sensitivity to the light environment. Photoreceptors of the cichlid fish, Metriaclima zebra, mix different pairs of opsins in retinal regions that view distinct backgrounds. The mixing of visual pigments increases absorbance of the corresponding background, potentially aiding the detection of dark objects. Thus, opsin coexpression may be a novel mechanism of spectral tuning that could be useful for detecting prey, predators and mates. However, our calculations show that coexpression of some opsins can hinder colour discrimination, creating a trade-off between visual functions.     

Opsin sequences of the rod visual pigments in two species of Poeciliid fish

The rod opsin sequences from Gambusia affinis holbrooki and Poecilia reticulata were cloned and sequenced. The opsin sequences were found to be 96.8% identical, reflecting the similarity of the rod visual pigment absorbances in these two Poeciliid fish.     

The Eyes Have It: Regulatory and Structural Changes Both Underlie Cichlid Visual Pigment Diversity

A major goal of evolutionary biology is to unravel the molecular genetic mechanisms that underlie functional diversification and adaptation. We investigated how changes in gene regulation and coding sequence contribute to sensory diversification in two replicate radiations of cichlid fishes. In the clear waters of Lake Malawi, differential opsin expression generates diverse visual systems, with sensitivities extending from the ultraviolet to the red regions of the spectrum. These sensitivities fall into three distinct clusters and are correlated with foraging habits. In the turbid waters of Lake Victoria, visual sensitivity is constrained to longer wavelengths, and opsin expression is correlated with ambient light. In addition to regulatory changes, we found that the opsins coding for the shortest- and longest-wavelength visual pigments have elevated numbers of potentially functional substitutions. Thus, we present a model of sensory evolution in which both molecular genetic mechanisms work in concert. Changes in gene expression generate large shifts in visual pigment sensitivity across the collective opsin spectral range, but changes in coding sequence appear to fine-tune visual pigment sensitivity at the short- and long-wavelength ends of this range, where differential opsin expression can no longer extend visual pigment sensitivity.     

Bat Eyes Have Ultraviolet-Sensitive Cone Photoreceptors

Mammalian retinae have rod photoreceptors for night vision and cone photoreceptors for daylight and colour vision. For colour discrimination, most mammals possess two cone populations with two visual pigments (opsins) that have absorption maxima at short wavelengths (blue or ultraviolet light) and long wavelengths (green or red light). Microchiropteran bats, which use echolocation to navigate and forage in complete darkness, have long been considered to have pure rod retinae. Here we use opsin immunohistochemistry to show that two phyllostomid microbats, Glossophaga soricina and Carollia perspicillata, possess a significant population of cones and express two cone opsins, a shortwave-sensitive (S) opsin and a longwave-sensitive (L) opsin. A substantial population of cones expresses S opsin exclusively, whereas the other cones mostly coexpress L and S opsin. S opsin gene analysis suggests ultraviolet (UV, wavelengths <400 nm) sensitivity, and corneal electroretinogram recordings reveal an elevated sensitivity to UV light which is mediated by an S cone visual pigment. Therefore bats have retained the ancestral UV tuning of the S cone pigment. We conclude that bats have the prerequisite for daylight vision, dichromatic colour vision, and UV vision. For bats, the UV-sensitive cones may be advantageous for visual orientation at twilight, predator avoidance, and detection of UV-reflecting flowers for those that feed on nectar.     

All-trans-retinal forms a visible-absorbing pigment with human rod opsin

Rhodopsin activation elicits transmembrane currents due to electrostatic events associated with conformational changes. We employed the sensitive rhodopsin early receptor current approach to reevaluate whether all-trans-retinal can form a visual pigment with rod opsin apoprotein. An opsin shift above 440 nm is induced in the action spectrum of charge motions caused by visible flashes in cells expressing human rod opsin and regenerated with all-trans-retinal, compared to cells without opsin. Near-ultraviolet stimulation of opsin regenerated with all-trans-retinal promotes charge motions similar to those arising from the meta-II signaling state while photochemically regenerating a pigment with ground state charge motion properties. These results indicate that all-trans-retinal can form a visual pigment with opsin, through both protonated and unprotonated Schiff base linkages and likely within the native ligand binding pocket at lysine-296. The agonist effects of all-trans-retinal may relate to its structural accommodation within the core of opsin, similar to other G-protein-coupled receptors.     

Adaptive molecular evolution in the opsin genes of rapidly speciating cichlid species

Cichlid fish inhabit a diverse range of environments that vary in the spectral content of light available for vision. These differences should result in adaptive selective pressure on the genes involved in visual sensitivity, the opsin genes. This study examines the evidence for differential adaptive molecular evolution in East African cichlid opsin genes due to gross differences in environmental light conditions. First, we characterize the selective regime experienced by cichlid opsin genes using a likelihood ratio test format, comparing likelihood models with different constraints on the relative rates of amino acid substitution, across sites. Second, we compare turbid and clear lineages to determine if there is evidence of differences in relative rates of substitution. Third, we present evidence of functional diversification and its relationship to the photic environment among cichlid opsin genes. We report statistical evidence of positive selection in all cichlid opsin genes, except short wavelength-sensitive 1 and short wavelength-sensitive 2b. In all genes predicted to be under positive selection, except short wavelength-sensitive 2a, we find differences in selective pressure between turbid and clear lineages. Potential spectral tuning sites are variable among all cichlid opsin genes; however, patterns of substitution consistent with photic environment-driven evolution of opsin genes are observed only for short wavelength-sensitive 1 opsin genes. This study identifies a number of promising candidate-tuning sites for future study by site-directed mutagenesis. This work also begins to demonstrate the molecular evolutionary dynamics of cichlid visual sensitivity and its relationship to the photic environment.     

Rod opsin cDNA sequence from the sand goby (Pomatoschistus minutus) compared with those of other vertebrates.

The absorbance spectra of rods from the sand goby were measured by using microspectrophotometry. Analysis of the averaged spectra shows that the rod visual pigment has a maximum absorbance (lambda max) at approximately 501 nm. A sand goby retinal cDNA library was constructed and then screened with a partial sand goby rod opsin clone obtained by the polymerase chain reaction (PCR). The screening of the library yielded a full length rod opsin clone. The cDNA sequence and deduced amino acid sequence of this clone are compared with those of other vertebrate rod opsins.     

Retinal Cone Photoreceptors of the Deer Mouse Peromyscus maniculatus: Development,Topography, Opsin Expression and Spectral Tuning

A quantitative analysis of photoreceptor properties was performed in the retina of the nocturnal deer mouse, Peromyscus maniculatus, using pigmented (wildtype) and albino animals. The aim was to establish whether the deer mouse is a more suitable model species than the house mouse for photoreceptor studies, and whether oculocutaneous albinism affects its photoreceptor properties. In retinal flatmounts, cone photoreceptors were identified by opsin immunostaining, and their numbers, spectral types, and distributions across the retina were determined. Rod photoreceptors were counted using differential interference contrast microscopy. Pigmented P. maniculatus have a rod-dominated retina with rod densities of about 450.000/mm² and cone densities of 3000 - 6500/mm². Two cone opsins, shortwave sensitive (S) and middle-to-longwave sensitive (M), are present and expressed in distinct cone types. Partial sequencing of the S opsin gene strongly supports UV sensitivity of the S cone visual pigment. The S cones constitute a 5-15% minority of the cones. Different from house mouse, S and M cone distributions do not have dorsoventral gradients, and coexpression of both opsins in single cones is exceptional (<2% of the cones). In albino P. maniculatus, rod densities are reduced by approximately 40% (270.000/mm²). Overall, cone density and the density of cones exclusively expressing S opsin are not significantly different from pigmented P. maniculatus. However, in albino retinas S opsin is coexpressed with M opsin in 60-90% of the cones and therefore the population of cones expressing only M opsin is significantly reduced to 5-25%. In conclusion, deer mouse cone properties largely conform to the general mammalian pattern, hence the deer mouse may be better suited than the house mouse for the study of certain basic cone properties, including the effects of albinism on cone opsin expression.     

Spectral tuning in salamander visual pigments studied with dihydroretinal chromophores.

In visual pigments, opsin proteins regulate the spectral absorption of a retinal chromophore by mechanisms that change the energy level of the excited electronic state relative to the ground state. We have studied these mechanisms by using photocurrent recording to measure the spectral sensitivities of individual red rods and red (long-wavelength-sensitive) and blue (short-wavelength-sensitive) cones of salamander before and after replacing the native 3-dehydro 11-cis retinal chromophore with retinal analogs: 11-cis retinal, 3-dehydro 9-cis retinal, 9-cis retinal, and 5,6-dihydro 9-cis retinal. The protonated Schiff's bases of analogs with unsaturated bonds in the ring had broader spectra than the same chromophores bound to opsins. Saturation of the bonds in the ring reduced the spectral bandwidths of the protonated Schiff's bases and the opsin-bound chromophores and made them similar to each other. This indicates that torsion of the ring produces spectral broadening and that torsion is limited by opsin. Saturating the 5,6 double bond in retinal reduced the perturbation of the chromophore by opsin in red and in blue cones but not in red rods. Thus an interaction between opsin and the chromophoric ring shifts the spectral maxima of the red and blue cone pigments, but not that of the red rod pigment.     

Occupancy of the chromophore binding site of opsin activates visual transduction in rod photoreceptors

The retinal analogue beta-ionone was used to investigate possible physiological effects of the noncovalent interaction between rod opsin and its chromophore 11-cis retinal. Isolated salamander rod photoreceptors were exposed to bright light that bleached a significant fraction of their pigment, were allowed to recover to a steady state, and then were exposed to beta-ionone. Our experiments show that in bleach-adapted rods beta-ionone causes a decrease in light sensitivity and dark current and an acceleration of the dim flash photoresponse and the rate constants of guanylyl cyclase and cGMP phosphodiesterase. Together, these observations indicate that in bleach-adapted rods beta-ionone activates phototransduction in the dark. Control experiments showed no effect of beta-ionone in either fully dark-adapted or background light-adapted cells, indicating direct interaction of beta-ionone with the free opsin produced by bleaching. We speculate that beta-ionone binds specifically in the chromophore pocket of opsin to produce a complex that is more catalytically potent than free opsin alone. We hypothesize that a similar reaction may occur in the intact retina during pigment regeneration. We propose a model of rod pigment regeneration in which binding of 11-cis retinal to opsin leads to activation of the complex accompanied by a decrease in light sensitivity. The subsequent covalent attachment of retinal to opsin completely inactivates opsin and leads to the recovery of sensitivity. Our findings resolve the conflict between biochemical and physiological data concerning the effect of the occupancy of the chromophore binding site on the catalytic potency of opsin. We show that binding of beta-ionone to rod opsin produces effects opposite to its previously described effects on cone opsin. We propose that this distinction is due to a fundamental difference in the interaction of rod and cone opsins with retinal, which may have implications for the different physiology of the two types of photoreceptors.     

Immunohistochemical evidence for multiple photosystems in box jellyfish

Cubomedusae (box jellyfish) possess a remarkable visual system with 24 eyes distributed in four sensory structures termed rhopalia. Each rhopalium is equipped with six eyes: two pairs of pigment cup eyes and two unpaired lens eyes. Each eye type probably captures specific features of the visual environment. To investigate whether multiple types of photoreceptor cells are present in the rhopalium, and whether the different eye types possess different types of photoreceptors, we have used immunohistochemistry with a range of vertebrate opsin antibodies to label the photoreceptors, and electroretinograms (ERG) to determine their spectral sensitivity. All photoreceptor cells of the two lens eyes of the box jellyfish Tripedalia cystophora and Carybdea marsupialis displayed immunoreactivity for an antibody directed against the zebrafish ultraviolet (UV) opsin, but not against any of eight other rhodopsin or cone opsin antibodies tested. In neither of the two species were the pigment cup eyes immunoreactive for any of the opsin antibodies. ERG analysis of the Carybdea lower lens eyes demonstrated a single spectral sensitivity maximum at 485 nm suggesting the presence of a single opsin type. Our data demonstrate that the lens eyes of box jellyfish utilize a single opsin and are thus color-blind, and that there is probably a different photopigment in the pigment cup eyes. The results support our hypothesis that the lens eyes and the pigment cup eyes of box jellyfish are involved in different and specific visual tasks.