Although much is known about the visual system of vertebrates in general, studies regarding vision in reptiles, and snakes in particular, are scarce. Reptiles display diverse ocular structures, including different types of retinae such as pure cone, mostly rod, or duplex retinas (containing both rods and cones); however, the same five opsin-based photopigments are found in many of these animals. It is thought that ancestral snakes were nocturnal and/or fossorial, and, as such, they have lost two pigments, but retained three visual opsin classes. These are the RH1 gene (rod opsin or rhodopsin-like-1) expressed in rods and two cone opsins, namely LWS (long-wavelength-sensitive) and SWS1 (short-wavelength-sensitive-1) genes. Until recently, the study of snake photopigments has been largely ignored. However, its importance has become clear within the past few years as studies reconsider Walls’ transmutation theory, which was first proposed in the 1930s. In this study, the visual pigments of Bothrops atrox (the common lancehead), a South American pit viper, were examined. Specifically, full-length RH1 and LWS opsin gene sequences were cloned, as well as most of the SWS1 opsin gene. These sequences were subsequently used for phylogenetic analysis and to predict the wavelength of maximum absorbance (λmax) for each photopigment. This is the first report to support the potential for rudimentary color vision in a South American viper, specifically a species that is regarded as being nocturnal.

Unlike the mammalian retina, the teleost fish retina undergoes persistent neurogenesis from intrinsic stem cells. In marine teleosts, most cone photoreceptor genesis occurs early in the embryonic and larval stages, and rods are added primarily during and after metamorphosis. This study demonstrates a developmental paradigm in elopomorph fishes in which retinas are rod-dominated in larvae, but undergo periods of later cone genesis. Retinal characteristics were compared at different developmental stages among three ecologically distinct elopomorph fishes—ladyfish (Elops saurus), bonefish (Albula vulpes), and speckled worm eel (Myrophis punctatus). The objectives were to improve our understanding of (1) the developmental strategy in the elopomorph retina, (2) the functional architecture of the retina as it relates to ecology, and (3) how the light environment influences photoreceptor genesis. Photoreceptor morphologies, distributions, and spectral absorption were studied at larval, juvenile, and adult stages. Premetamorphic retinas in all three species are rod-dominated, but the retinas of these species undergo dramatic change over the course of development, resulting in juvenile and adult retinal characteristics that correlate closely with ecology. Adult E. saurus has high rod densities, grouped photoreceptors, a reflective tapetum, and longer-wavelength photopigments, supporting vision in turbid, low-light conditions. Adult A. vulpes has high cone densities, low rod densities, and shorter-wavelength photopigments, supporting diurnal vision in shallow, clear water. M. punctatus loses cones during metamorphosis, develops new cones after settlement, and maintains high rod but low cone densities, supporting primarily nocturnal vision. M. punctatus secondary cone genesis occurs rapidly throughout the retina, suggesting a novel mechanism of vertebrate photoreceptor genesis. Finally, in postsettlement M. punctatus, the continuous presence or absence of visible light modulates rod distribution but does not affect secondary cone genesis, suggesting some degree of developmental plasticity influenced by the light environment.

Our current understanding of the spectral sensitivities of the mysticete whale rod-based visual pigments is based on two species, the gray whale (Eschrichtius robustus) and the humpback whale (Megaptera novaeangliae) possessing absorbance maxima determined from difference spectra to be 492 and 497 nm, respectively. These absorbance maxima values are blueshifted relative to those from typical terrestrial mammals (≈500 nm) but are redshifted when compared to those identified in the odontocetes (479–484 nm). Although these mysticete species represent two of the four mysticete families, they do not fully represent the mysticete whales in terms of foraging strategy and underwater photic environments where foraging occurs. In order to better understand the spectral sensitivities of the mysticete whale rod visual pigments, we have examined the rod opsin genes from 11 mysticete species and their associated amino acid substitutions. Based on the amino acids occurring at positions 83, 292, and 299 along with the directly determined dark spectra from expressed odontocete and mysticete rod visual pigments, we have determined that the majority of mysticete whales possess deep-sea and pelagic like rod visual pigments with absorbance maxima between 479 and 484 nm. Finally, we have defined the five amino acid substitution events that determine the resulting absorbance spectra and associated absorbance maxima for the mysticete whale rod visual pigments examined here.

Vision is used in nearly all aspects of animal behavior, from prey and predator detection to mate selection and parental care. However, the light environment typically is not uniform in every direction, and visual tasks may be specific to particular parts of an animal’s field of view. These spatial differences may explain the presence of several adaptations in the eyes of vertebrates that alter spectral sensitivity of the eye in different directions. Mechanisms that alter spectral sensitivity across the retina include (but are not limited to) variations in: corneal filters, oil droplets, macula lutea, tapeta, chromophore ratios, photoreceptor classes, and opsin expression. The resultant variations in spectral sensitivity across the retina are referred to as intraretinal variability in spectral sensitivity (IVSS). At first considered an obscure and rare phenomenon, it is becoming clear that IVSS is widespread among all vertebrates, and examples have been found from every major group. This review will describe the mechanisms mediating differences in spectral sensitivity, which are in general well understood, as well as explore the functional significance of intraretinal variability, which for the most part is unclear at best.

The Atlantic tarpon, Megalops atlanticus, is a large piscivorous fish that supports economically important recreational fisheries in the Gulf of Mexico, Caribbean, and Florida Atlantic coast. Megalops atlanticus undergoes ontogenetic shifts in morphology, hatching in the open ocean as larvae (less than 1 cm in length), moving into hypoxic turbid mangrove marshes as juveniles (around 10 cm in length), and then moving into coastal oceanic waters as adults (over 100 cm in length). In this study, photoreceptor distributions, opsin distributions, and photoreceptor absorbance characteristics were studied with light microscopy, transmission electron microscopy, antiopsin immunofluorescence, and microspectrophotometry, respectively, at four ecologically distinct life-history stages—premetamorphic larva, settlement stage, juvenile, and adult. The purposes of this study were 1) to determine the extent to which the retina of M. atlanticus changes over the course of development and 2) to relate these retinal changes with ecological shifts between developmental stages. The new data presented here indicate that the M. atlanticus retina changes substantially in rod and cone distributions and absorbance characteristics over the course of development and that these changes correlate closely with those in habitat and behavior. We show that M. atlanticus has a rod-dominated retina at the larval stage (which is unusual for teleost larvae) and that the scotopic visual system becomes far better developed with maturity, adding a substantial tapetum and high densities of small, bundled, and stacked rod cells. We also show that there are shifts in cone and rod spectral sensitivities and an increase in the diversity of spectrally distinct cone classes, including the addition of ultraviolet cones as fish mature into adults.

In zebrafish, the first class of cone photoreceptor to become morphologically distinct is the ultraviolet-sensitive short single cone, at 4 days postfertilization, whereas the last class, the red- and green-sensitive double cone, becomes distinct at 10 days postfertilization. We have examined the time course of visual pigment gene expression in zebrafish using whole-mount in situ hybridization. Within the retina, opsins may be detected as early as 40 h postfertilization with the ultraviolet and rod visual pigments being expressed before the blue- (48 h) and red- (60 h) sensitive pigments. In the pineal, red-sensitive opsin is expressed at 48 h postfertilization. Visual pigment expression provides a useful tool for investigations of early cell fate in zebrafish.

The retinas of placental mammals appear to lack the large number and morphological diversity of cone subtypes found in diurnal reptiles. We have now studied the photoreceptor layer of a South American marsupial (Didelphis marsupialis aurita) by peanut agglutinin labeling of the cone sheath and by labeling of cone outer segments with monoclonal anti-visual pigment antibodies that have been proven to consistently label middle-to-long wavelength (COS-1) and short-wavelength (OS-2) cone subpopulations in placental mammals. Besides a dominant rod population (max. = 400,000/mm2) four subtypes of cones (max. = 3000/mm2) were identified. The outer segments of three cone subtypes were labeled by COS-1: a double cone with a principal cone containing a colorless oil droplet, a single cone with oil droplet, and another single cone. A second group of single cones lacking oil droplets was labeled by OS-2 antibody. The topography of these cone subtypes showed striking anisotropies. The COS-1 labeled single cones without oil droplets were found all over the retina and constituted the dominant population in the area centralis located in the temporal quadrant of the upper, tapetal hemisphere. The population of OS-2 labeled cones was also ubiquitous although slightly higher in the upper hemisphere (200/mm2). The COS-1 labeled cones bearing an oil droplet, including the principal member of double cones, were concentrated (800/mm2) in the inferior, non-tapetal half of the retina. The two spectral types of single cones resemble those of dichromatic photopic systems in most placental mammals. The additional set of COS-1 labeled cones is a distinct marsupial feature. The presence of oil droplets in this cone subpopulation, its absence in the area centralis, and the correlation with the non-tapetal inferior hemisphere suggest a functional specialization, possibly for mesopic conditions. Thus, sauropsid features have been retained but probably with a modified function.

Stomatopod crustaceans possess apposition compound eyes that contain more photoreceptor types than any other animal described. While the anatomy and physiology of this complexity have been studied for more than two decades, few studies have investigated the molecular aspects underlying the stomatopod visual complexity. Based on previous studies of the structure and function of the different types of photoreceptors, stomatopod retinas are hypothesized to contain up to 16 different visual pigments, with 6 of these having sensitivity to middle or long wavelengths of light. We investigated stomatopod middle- and long-wavelength-sensitive opsin genes from five species with the hypothesis that each species investigated would express up to six different opsin genes. In order to understand the evolution of this class of stomatopod opsins, we examined the complement of expressed transcripts in the retinas of species representing a broad taxonomic range (four families and three superfamilies). A total of 54 unique retinal opsins were isolated, resulting in 6–15 different expressed transcripts in each species. Phylogenetically, these transcripts form six distinct clades, grouping with other crustacean opsins and sister to insect long-wavelength visual pigments. Within these stomatopod opsin groups, intra- and interspecific clusters of highly similar transcripts suggest that there has been rampant recent gene duplication. Some of the observed molecular diversity is also due to ancient gene duplication events within the stem crustacean lineage. Using evolutionary trace analysis, 10 amino acid sites were identified as functionally divergent among the six stomatopod opsin clades. These sites form tight clusters in two regions of the opsin protein known to be functionally important: six in the chromophore-binding pocket and four at the cytoplasmic surface in loops II and III. These two clusters of sites indicate that stomatopod opsins have diverged with respect to both spectral tuning and signal transduction.

The abundance and spatial distribution of retinal cone photoreceptors change during thyroid hormone (TH)-induced and natural development of rainbow trout (Oncorhynchus mykiss). These changes are thought to allow the fish to adapt to different photic environments throughout its life history. To date, the ontogeny of rainbow trout cone photoreceptors has been examined using physiological and morphological approaches. In this study, we extended these observations by measuring opsin gene expression in retinal quadrants during natural and TH-induced development. Gene expression during natural development was investigated in retinae from fish at both parr and smolt stages. The role of TH in modulating opsin gene expression was determined in TH-treated parr and control fish sampled after two, nine, and 22 days of treatment. Total RNA was isolated from each retinal quadrant and steady-state opsin mRNA levels were measured using reverse transcriptase real-time quantitative polymerase chain reaction (QPCR) analysis. Expression of ultraviolet-sensitive opsin (SWS1), rod opsin (RH1), middle wavelength-sensitive opsin (RH2), and long wavelength-sensitive opsin (LWS) transcripts vary spatially in the parr retina. Smolts, compared to parr, had downregulated SWS1 expression in all quadrants, lower LWS expression dorsally, higher RH1 expression nasally, and higher RH2 expression dorsally. In TH-treated parr, SWS1 opsin expression was downregulated in the nasal quadrants by two days. SWS1 displayed the greatest degree of downregulation in all quadrants after nine days of treatment, with an increase in short wavelength-sensitive (SWS2) and RH2 opsin mRNA expression in the temporal quadrants. This study reveals that opsin genes display spatially significant differences within rainbow trout retina in their level of mRNA expression, and that regulation of opsin expression is a dynamic process that is influenced by TH. This is particularly evident for SWS1 gene expression in parr following TH-induced and natural development.

The biochemical and morphological specializations of rod and cone photoreceptors reflect their roles in sight. The apoprotein opsin, which converts photons into chemical signals, functions at one end of these highly polarized cells, in the outer segment. Previous work has shown that the mRNA of rod opsin, the opsin specific to rods, is renewed in the outer segment with a diurnal rhythm in the retina of the teleost fish Haplochromis burtoni. Here we show that in the same species, all three cone opsin mRNAs (blue, green, and red) also have a diurnal rhythm of expression. Quantitative real-time polymerase chain reaction (PCR) with primer pairs specific for the cone photoreceptor opsin subtypes was used to detect opsin mRNA abundance in animals sacrificed at 3-h intervals around the clock. All three cone opsins were expressed with diurnal rhythms similar to each other but out of phase with the rod opsin rhythm. Specifically, cone opsin expression occurs at a higher level near the onset of the dark period, when cones are not used for vision. Finally, we found that the rhythm of cone opsin expression in fish appears to be light dependent, as prolonged darkness changes normal diurnal expression patterns.

Most molecular studies on the visual system in fish have been performed on freshwater teleosts such as goldfish and zebrafish where cones and rods appear simultaneously. Many marine fishes have long larval phase in the upper pelagic zone before transformation into a juvenile and a benthic life style. The retina at the larval stages consists of only single cone cells; later during metamorphosis double cones and rods develop. The flatfish Atlantic halibut (Hippoglossus hippoglossus) is a typical example of a marine species with such a two-step retina development. In this study, we have cloned five different opsins from Atlantic halibut larvae and juvenile retinas. Sequence comparisons with other opsins and phylogenetic analysis show that the five genes belong to the opsins of long-wavelength sensitive (L); middle-wavelength sensitive, MCone and MRod; and short-wavelength sensitive, SBlue and SUltraviolet, respectively. In situ hybridization analysis reveals expression in double cone (L and MCone), single cone (SBlue and SUltraviolet), and rod (MRod) types of photoreceptor cells in juvenile halibut retina. The visual system in Atlantic halibut seems therefore to have all four types of cone photoreceptors in addition to rod photoreceptors. This work shows for the first time molecular isolation of a complete set of retinal visual pigment genes from a marine teleost and describes the first cloning of an ultraviolet-sensitive opsin type from a marine teleost.

The visual pigment from the ultraviolet (UV) cone photoreceptor of the tiger salamander has been cloned, expressed, and characterized. The cDNA contains a full-length open reading frame encoding 347 amino acids. The phylogenetic analysis indicates that the highest sequence homology is to the visual pigments in the S group. The UV opsin was tagged at the carboxy-terminus with the sequence for the 1D4 epitope. This fusion opsin was expressed in COS-1 cells, regenerated with 11-cis retinal (A1) and immuno-purified, yielding a pigment with an absorbance maximum (λmax) of 356 nm which is blue shifted from the absorption of retinal itself. The transducin activation assay demonstrated that this pigment is able to activate rod transducin in a light-dependent manner. Regeneration with 11-cis 3,4-dehydroretinal (A2) yielded a pigment with a λmax of 360 nm, only 4 nm red shifted from that of the A1 pigment, while bovine rhodopsin generated with A2 showed a 16-nm red shift from the corresponding A1 pigment. These results demonstrate that the trend for a shorter wavelength pigment to have a smaller shift of λmax between the A1 and A2 pigments also fits UV pigments. We hypothesize that the small red shift with A2 could be due to a twist in the chromophore that essentially isolates the ring double bond(s) from conjugation with the rest of the polyene chain.

The present study has examined the spatial and temporal expression patterns of various proteins associated with the structure and function of mature photoreceptor outer segments in the developing ferret's retina using immunocytochemistry and RT-PCR. One set of proteins, including rod opsin, arrestin, and recoverin, was detected progressively in photoreceptors as they became postmitotic, being expressed well before the differentiation of outer segments. A second set of proteins, including β- and γ-transducin, cGMP-phosphodiesterase, phosducin, rhodopsin kinase, rod cGMP-gated cation channel protein, and peripherin, displayed a contrasting temporal onset and pattern of spatial emergence. These latter proteins first became detectable either shortly before or coincident with outer segment formation, and were expressed simultaneously in both older and younger photoreceptor cells. A third set, the short wavelength-sensitive (SWS) and medium wavelength-sensitive (MWS) cone opsin proteins, was the last to be detected, but materialized in a spatio-temporal pattern reminiscent of the neurogenetic gradient of the cones. These different spatial and temporal patterns indicate that cellular maturation must play a primary role in regulating the onset of expression of some of these proteins, while extrinsic signals must act to coordinate the expression of other proteins across photoreceptors of different ages.

Adult teleost fish retinas can regenerate neurons following either surgical or pharmacological injury. The cellular milieu of the damaged retina within which regenerated neurons are produced might be different in these two model systems of retinal injury, and thus the phenotypic attributes of regenerated neurons in the two model systems might also differ. To determine if the phenotypic attributes of photoreceptors, and by extension the recovery of vision, are different between these two model systems, we compared the visual pigment content and morphology of photoreceptors derived from goldfish retinas of both models with control retina. Visual pigments—which consist of a protein moiety (opsin) and a chromophore—were analyzed in single, isolated photoreceptors using microspectrophotometric techniques. We report that visual pigments and photoreceptor morphologies in the surgical model closely matched those of native retina. In contrast, neither photoreceptor morphology nor visual pigment content matched closely in the pharmacological model. The results indicate that phenotypic attributes of photoreceptors can differ significantly between the two model systems of retinal regeneration, but that in both systems, rod- and cone-mediated visual functions can potentially be reestablished.

Adult Japanese quail have an endogenous circadian clock located in their eyes that has been shown to regulate melatonin biosynthesis. We investigated if a circadian oscillator is present in cultures of dispersed embryonic quail retina. Melatonin release in retinal cell culture is modulated by the light cycle, indicating that there are functional photoreceptors in culture. However, when cultures were placed in constant darkness no rhythm of melatonin was observed, indicating that at this period of development the circadian oscillator does not influence melatonin release. To explore further the question of whether a circadian oscillator is present in embryonic cell culture, we examined expression of iodopsin, the red visual pigment. Iodopsin mRNA is expressed in a circadian rhythm with peak levels occurring late in the afternoon (ZT 9). Analysis indicates that the clock influence is at the level of gene transcription. These results suggest that a clock is not “hooked up” to melatonin release embryonically or that a different oscillator regulates photopigment expression versus melatonin release.

We report here a reexamination of the developmental expression of cone opsins in the zebrafish retina. The red- and blue-sensitive opsins appear at 51 h postfertilization (hpf) whereas ultraviolet (UV) opsin is not seen until after 55 hpf. More cells show red cone opsin expression than blue at 51 and 55 hpf, indicating the sequence of cone opsin expression in zebrafish is first red, then blue, and finally UV. Curiously, morphological development of the cones is in reverse order; UV cones appear quite mature by day 6–7 postfertilization (pf), but morphologically, red cones do not appear adult-like until 15–20 days pf.

Zebrafish (Danio rerio) represents an excellent genetic model for vertebrate visual system studies. Because the opsin proteins are ideal markers of specific photoreceptor cell types, we cloned six different zebrafish opsin cDNAs. Based on pairwise alignments and phylogenetic comparisons between the predicted zebrafish opsin amino acid sequences and other vertebrate opsins, the cDNAs encode rhodopsin, two different green opsins (zfgr1 and zfgr2), a red, a blue, and an ultraviolet opsin. Phylogenetic analysis indicates the zfgr1 protein occupies a well-resolved dendrogram branch separate from the other green opsins examined, while zebrafish ultraviolet opsin is closely related to the human blue- and chicken violet-sensitive proteins. Polyclonal antisera were generated against individual bacterial fusion proteins containing either the red, blue, or ultraviolet amino termini or the rod or green opsin carboxyl termini. Immunolocalization on adult zebrafish frozen sections demonstrates the green and red opsins are each expressed in different members of the double cone cell pair, the blue opsin is detected in long single cones, and the ultraviolet opsin protein is expressed in the short single cones. In 120-h postfertilization wholemounts, green, red, blue, and ultraviolet opsin-positive cells are detected in an orderly arrangement throughout the entire retina. The antibodies' photoreceptor-type specificity indicates they will be useful for characterizing both wild-type and mutant zebrafish retinas.

The neotenic tiger salamander retina is a major model system for the study of retinal physiology and circuitry, yet there are unresolved issues regarding the organization of the photoreceptors and the photoreceptor mosaic. The rod and cone subtypes in the salamander retina were identified using a combination of morphological and immunocytochemical markers for specific rod and cone opsin epitopes. Because the visual pigment mechanisms present in the tiger salamander retina are well characterized and the antibodies employed in these studies are specific for particular rod and cone opsin epitopes, we also were able to identify the spectral class of the various rod and cone subtypes. Two classes of rods corresponding to the “red” and “green” rods previously reported in amphibian retinas were identified. In serial semithin section analyses, rods and cones comprised 62.4 ± 1.4% and 37.6 ± 1.4% of all photoreceptors, respectively. One rod type comprising 98.0 ± 0.7% of all rods showed the immunological and morphological characteristics of “red” rods, which are maximally sensitive to middle wavelengths. The second rod subtype comprised 2.0 ± 0.7% of all rods and possessed the immunological and morphological characteristics of “green” rods, which are maximally sensitive to short wavelengths. By morphology four cone types were identified, showing three distinct immunological signatures. Most cones (84.8 ± 1.5% of all cones), including most large single cones, the accessory and principal members of the double cone, and some small single cones, showed immunolabeling by antisera that recognize long wavelength-sensitive cone opsins. A subpopulation of small single cones (8.4 ± 1.7% of all cones) showed immunolabeling for short wavelength-sensitive cone opsin. A separate subpopulation of single cones which included both large and small types (6.8 ± 1.4% of all cones) was identified as the UV-Cone population and showed immunolabeling by antibodies that recognize rod opsin epitopes. Analysis of flatmounted retinas yielded similar results. All photoreceptor types appeared to be distributed in all retinal regions. There was no obvious crystalline organization of the various photoreceptor subtypes in the photoreceptor mosaic.

The distributions of rod and cone photoreceptors have been determined in the retina of the California ground squirrel, Spermophilus beecheyi. Retinas were fixed by perfusion and the rods and cones were detected with indirect immunofluorescence using opsin antibodies. Local densities were determined at 2-mm intervals across the entire retina, from which total numbers of each receptor type were estimated and isodensity distributions were constructed. The ground squirrel retina contains 7.5 million cones and 1.27 million rods. The peak density for the cones (49,550/mm2) is found in a horizontal strip of central retina 2 mm ventral to the elongated optic nerve head, falling gradually to half this value in the dorsal and ventral retinal periphery. Of the cones, there are 14 M cones for every S cone. S cone density is relatively flat across most of the retina, reaching a peak (4500/mm2) at the temporal end of the visual streak. There is one exception to this, however: S cone density climbs dramatically at the extreme dorso-nasal retinal margin (20,000/mm2), where the local ratio of S to M cones equals 1. Rod density is lowest in the visual streak, where the rods comprise less than 5% of the local photoreceptor population, increasing conspicuously in the ventral retina, where the rods achieve 30% of the local photoreceptor population (13,000/mm2). The functional importance of the change in S to M cone ratio at the dorsal circumference of the retina is compromised by the extremely limited portion of the visual field subserved by this retinal region. The significance for vision, if any, remains to be determined. By contrast, the change in rod/cone ratio between the dorsal and ventral halves of the retina indicates a conspicuous asymmetry in the ground squirrel's visual system, suggesting a specialization for maximizing visual sensitivity under dim levels of illumination in the superior visual field.