RH2 Opsin Research Articles

Differential sensitivity to estrogen-induced opsin expression in two poeciliid freshwater fish species

The sensory system shapes an individual’s perception of the world, including social interactions with conspecifics, habitat selection, predator detection, and foraging behavior. Sensory signaling can be modulated by steroid hormones, making these processes particularly vulnerable to environmental perturbations. Here we examine the influence of exogenous estrogen manipulation on the visual physiology of female western mosquitofish (Gambusia affinis) and sailfin mollies (Poecilia latipinna), two poeciliid species that inhabit freshwater environments across the southern United States. We conducted two experiments to address this aim. First, we exposed females from both species to a one-week dose response experiment with three treatments of waterborne β-estradiol. Next, we conducted a one-week estrogen manipulation experiment with a waterborne estrogen (β-Estradiol), a selective estrogen receptor modulator (tamoxifen), or combination estrogen and tamoxifen treatment. We used quantitative PCR (qPCR) to examine the expression of cone opsins (SWS1, SWS2b, SWS2a, Rh2, LWS), rhodopsin (Rh1), and steroid receptor genes (ARα, ARβ, ERα, ERβ2, GPER) in the eyes of individual females from each species. Results from the dose response experiment revealed estradiol-sensitivity in opsin (SWS2a, Rh2, Rh1) and androgen receptor (ARα, ARβ) gene expression in mosquitofish females, but not sailfins. Meanwhile, our estrogen receptor modulation experiments revealed estrogen sensitivity in LWS opsin expression in both species, along with sensitivity in SWS1, SWS2b, and Rh2 opsins in mosquitofish. Comparisons of control females across experiments reveal species-level differences in opsin expression, with mosquitofish retinas dominated by short-wavelength sensitive opsins (SWS2b) and sailfins retinas dominated by medium- and long-wavelength sensitive opsins (Rh2 and LWS). Our research suggests that variation in exogenous levels of sex hormones within freshwater environments can modify the visual physiology of fishes in a species-specific manner.

South American Weakly Electric Fish (Gymnotiformes) Are Long-Wavelength-Sensitive Cone Monochromats

Losses of cone opsin genes are noted in animals that are nocturnal or rely on senses other than vision. We investigated the cone opsin repertoire of night-active South American weakly electric fish. We obtained opsin gene sequences from genomic DNA of 3 gymnotiforms (Eigenmannia virescens, Sternopygus macrurus, Apteronotus albifrons) and the assembled genome of the electric eel (Electrophorus electricus). We identified genes for long-wavelength-sensitive (LWS) and medium-wavelength-sensitive cone opsins (RH2) and rod opsins (RH1). Neither of the 2 short-wavelength-sensitive cone opsin genes were found and are presumed lost. The fact that Electrophorus has a complete repertoire of extraretinal opsin genes and conservation of synteny with the zebrafish (Danio rerio) for genes flanking the 2 short-wavelength-sensitive opsin genes supports the supposition of gene loss. With microspectrophotometry and electroretinograms we observed absorption spectra consistent with RH1 and LWS but not RH2 opsins in the retinal photoreceptors of E. virescens. This profile of opsin genes and their retinal expression is identical to the gymnotiform's sister group, the catfish, which are also nocturnally active and bear ampullary electroreceptors, suggesting that this pattern likely occurred in the common ancestor of gymnotiforms and catfish. Finally, we noted an unusual N-terminal motif lacking a conserved glycosylation consensus site in the RH2 opsin of gymnotiforms, a catfish and a characin (Astyanax mexicanus). Mutations at this site influence rhodopsin trafficking in mammalian photoreceptors and cause retinitis pigmentosa. We speculate that this unusual N terminus may be related to the absence of the RH2 opsin in the cones of gymnotiforms and catfish.

From crypsis to mimicry: changes in colour and the configuration of the visual system during ontogenetic habitat transitions in a coral reef fish.

Animals often change their habitat throughout ontogeny; yet, the triggers for habitat transitions and how these correlate with developmental changes - e.g. physiological, morphological and behavioural - remain largely unknown. Here, we investigated how ontogenetic changes in body coloration and of the visual system relate to habitat transitions in a coral reef fish. Adult dusky dottybacks, Pseudochromis fuscus, are aggressive mimics that change colour to imitate various fishes in their surroundings; however, little is known about the early life stages of this fish. Using a developmental time series in combination with the examination of wild-caught specimens, we revealed that dottybacks change colour twice during development: (i) nearly translucent cryptic pelagic larvae change to a grey camouflage coloration when settling on coral reefs; and (ii) juveniles change to mimic yellow- or brown-coloured fishes when reaching a size capable of consuming juvenile fish prey. Moreover, microspectrophotometric (MSP) and quantitative real-time PCR (qRT-PCR) experiments show developmental changes of the dottyback visual system, including the use of a novel adult-specific visual gene (RH2 opsin). This gene is likely to be co-expressed with other visual pigments to form broad spectral sensitivities that cover the medium-wavelength part of the visible spectrum. Surprisingly, the visual modifications precede changes in habitat and colour, possibly because dottybacks need to first acquire the appropriate visual performance before transitioning into novel life stages.

Spatially differentiated expression of quadruplicated green-sensitive RH2 opsin genes in zebrafish is determined by proximal regulatory regions and gene order to the locus control region.

BackgroundFish are remarkably diverse in repertoires of visual opsins by gene duplications. Differentiation of their spatiotemporal expression patterns and absorption spectra enables fine-tuning of feature detection in spectrally distinct regions of the visual field during ontogeny. Zebrafish have quadruplicated green-sensitive (RH2) opsin genes in tandem (RH2-1, −2, −3, −4), which are expressed in the short member of the double cones (SDC). The shortest wavelength RH2 subtype (RH2-1) is expressed in the central to dorsal area of the adult retina. The second shortest wave subtype (RH2-2) is expressed overlapping with RH2-1 but extending outside of it. The second longest wave subtype (RH2-3) is expressed surrounding the RH2–2 area, and the longest wave subtype (RH2-4) is expressed outside of the RH2-3 area broadly occupying the ventral area. Expression of the four RH2 genes in SDC requires a single enhancer (RH2-LCR), but the mechanism of their spatial differentiation remains elusive.ResultsFunctional comparison of the RH2-LCR with its counterpart in medaka revealed that the regulatory role of the RH2-LCR in SDC-specific expression is evolutionarily conserved. By combining the RH2-LCR and the proximal upstream region of each RH2 gene with fluorescent protein reporters, we show that the RH2-LCR and the RH2-3 proximal regulatory region confer no spatial selectivity of expression in the retina. But those of RH2-1, −2 and −4 are capable of inducing spatial differentiation of expression. Furthermore, by analyzing transgenic fish with a series of arrays consisting of the RH2-LCR and multiple upstream regions of the RH2 genes in different orders, we show that a gene expression pattern related to an upstream region is greatly influenced by another flanking upstream region in a relative position-dependent manner.ConclusionsThe zebrafish RH2 genes except RH2-3 acquired differential cis-elements in the proximal upstream regions to specify the differential expression patterns. The input from these proximal elements collectively dictates the actual gene expression pattern of the locus, context-dependently. Importantly, competition for the RH2-LCR activity among the replicates is critical in this collective regulation, facilitating differentiation of expression among them. This combination of specificity and generality enables seemingly complicated spatial differentiation of duplicated opsin genes characteristic in fish.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0288-7) contains supplementary material, which is available to authorized users.

Sequence analysis and expression patterns of opsin genes in the longtooth grouper Epinephelus bruneus

Here, to evaluate ontogenic changes in visual sensitivity in juvenile longtooth grouper Epinephelus bruneus, we investigated opsin gene sequences and expression in larvae and juveniles. The longtooth grouper has 10 opsin genes: Rh1, five Rh2 genes (Rh2A1-1, Rh2A1-2, Rh2A2, g6738, and g6740), SWS1, two SWS2 genes (SWS2A1 and SWS2A2), and LWS. To date, five or six Rh2 opsin genes have been reported in perciform fishes. Differential opsin gene expression was examined by RT-PCR; the results suggested that switching from SWS1 to SWS2 occurs during early development, and that there is an absence of LWS expression between 14 and 40 days post-hatch. These ontogenetic changes in opsin gene expression may correspond to different requirements at different life stages.

Gene loss, adaptive evolution and the co-evolution of plumage coloration genes with opsins in birds.

BackgroundThe wide range of complex photic systems observed in birds exemplifies one of their key evolutionary adaptions, a well-developed visual system. However, genomic approaches have yet to be used to disentangle the evolutionary mechanisms that govern evolution of avian visual systems.ResultsWe performed comparative genomic analyses across 48 avian genomes that span extant bird phylogenetic diversity to assess evolutionary changes in the 17 representatives of the opsin gene family and five plumage coloration genes. Our analyses suggest modern birds have maintained a repertoire of up to 15 opsins. Synteny analyses indicate that PARA and PARIE pineal opsins were lost, probably in conjunction with the degeneration of the parietal organ. Eleven of the 15 avian opsins evolved in a non-neutral pattern, confirming the adaptive importance of vision in birds. Visual conopsins sw1, sw2 and lw evolved under negative selection, while the dim-light RH1 photopigment diversified. The evolutionary patterns of sw1 and of violet/ultraviolet sensitivity in birds suggest that avian ancestors had violet-sensitive vision. Additionally, we demonstrate an adaptive association between the RH2 opsin and the MC1R plumage color gene, suggesting that plumage coloration has been photic mediated. At the intra-avian level we observed some unique adaptive patterns. For example, barn owl showed early signs of pseudogenization in RH2, perhaps in response to nocturnal behavior, and penguins had amino acid deletions in RH2 sites responsible for the red shift and retinal binding. These patterns in the barn owl and penguins were convergent with adaptive strategies in nocturnal and aquatic mammals, respectively.ConclusionsWe conclude that birds have evolved diverse opsin adaptations through gene loss, adaptive selection and coevolution with plumage coloration, and that differentiated selective patterns at the species level suggest novel photic pressures to influence evolutionary patterns of more-recent lineages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1924-3) contains supplementary material, which is available to authorized users.

Evolutionary dynamics of Rh2 opsins in birds demonstrate an episode of accelerated evolution in the New World warblers (Setophaga).

Low rates of sequence evolution associated with purifying selection can be interrupted by episodic changes in selective regimes. Visual pigments are a unique system in which we can investigate the functional consequences of genetic changes, therefore connecting genotype to phenotype in the context of natural and sexual selection pressures. We study the RH2 and RH1 visual pigments (opsins) across 22 bird species belonging to two ecologically convergent clades, the New World warblers (Parulidae) and Old World warblers (Phylloscopidae) and evaluate rates of evolution in these clades along with data from 21 additional species. We demonstrate generally slow evolution of these opsins: both Rh1 and Rh2 are highly conserved across Old World and New World warblers. However, Rh2 underwent a burst of evolution within the New World genus Setophaga, where it accumulated substitutions at 6 amino acid sites across the species we studied. Evolutionary analyses revealed a significant increase in dN /dS in Setophaga, implying relatively strong selective pressures to overcome long-standing purifying selection. We studied the effects of each substitution on spectral tuning and found they do not cause large spectral shifts. Thus, substitutions may reflect other aspects of opsin function, such as those affecting photosensitivity and/or dark-light adaptation. Although it is unclear what these alterations mean for colour perception, we suggest that rapid evolution is linked to sexual selection, given the exceptional plumage colour diversification in Setophaga.

Spectral sensitivity of cone photoreceptors and opsin expression in two colour-divergent lineages of the lizard Ctenophorus decresii.

Intraspecific differences in sensory perception are rarely reported but may occur when a species range extends across varying sensory environments, or there is coevolution between the sensory system and a varying signal. Examples in colour vision and colour signals are rare in terrestrial systems. The tawny dragon lizard Ctenophorus decresii is a promising candidate for such intraspecific variation, because the species comprises two geographically and genetically distinct lineages in which throat colour (a social signal used in intra- and inter-specific interactions) is locally adapted to the habitat and differs between lineages. Male lizards from the southern lineage have UV-blue throats, whereas males from the northern lineage are polymorphic with four discrete throat colours that all show minimal UV reflectance. Here, we determine the cone photoreceptor spectral sensitivities and opsin expression of the two lineages, to test whether they differ, particularly in the UV wavelengths. Using microspectrophotometry on retinal cone photoreceptors, we identified a long-wavelength-sensitive (LWS) visual pigment, a 'short' and 'long' medium-wavelength-sensitive (MWS) pigment and a short-wavelength-sensitive (SWS) pigment, all of which did not differ in λmax between lineages. Through transcriptome analysis of opsin genes we found that both lineages express four cone opsin genes, including the SWS1 opsin with peak sensitivity in the UV range, and that amino acid sequences did not differ between lineages with the exception of a single leucine to valine substitution in the RH2 opsin. Counts of yellow and transparent oil droplets associated with LWS+MWS and SWS+UVS cones, respectively, showed no difference in relative cone proportions between lineages. Therefore, contrary to predictions, we find no evidence of differences between lineages in single cone photoreceptor spectral sensitivity or opsin expression. However, we confirm the presence of four single cone classes, suggesting tetrachromacy in C. decresii, and we also provide the first evidence of UV sensitivity in agamid lizards.

Molecular evidence that only two opsin subfamilies, the blue light- (SWS2) and green light-sensitive (RH2), drive color vision in Atlantic cod (Gadus morhua).

Teleosts show a great variety in visual opsin complement, due to both gene duplication and gene loss. The repertoire ranges from one subfamily of visual opsins (scotopic vision) including rod opsin only retinas seen in many deep-sea species to multiple subfamilies of visual opsins in some pelagic species. We have investigated the opsin repertoire of Atlantic cod (Gadus morhua) using information in the recently sequenced cod genome and found that despite cod not being a deep sea species it lacks visual subfamilies sensitive towards the most extreme parts of the light spectra representing UV and red light. Furthermore, we find that Atlantic cod has duplicated paralogs of both blue-sensitive SWS2 and green-sensitive RH2 subfamilies, with members belonging to each subfamily linked in tandem within the genome (two SWS2-, and three RH2A genes, respectively). The presence of multiple cone opsin genes indicates that there have been duplication events in the cod ancestor SWS2 and RH2 opsins producing paralogs that have been retained in Atlantic. Our results are supported by expressional analysis of cone opsins, which further revealed an ontogenetic change in the array of cone opsins expressed. These findings suggest life stage specific programs for opsin regulation which could be linked to habitat changes and available light as the larvae is transformed into an early juvenile. Altogether we provide the first molecular evidence for color vision driven by only two families of cone opsins due to gene loss in a teleost.

ALL THE BETTER TO SEE YOU WITH: PIGMENT VARIATIONS IN PARROTS

Birds around the world rely on colourful plumage to identify and assess their kinsmen. Birds see colour using four colour visual pigments in their cone cells in addition to a rod pigment that is used in dim light vision. Each of these visual pigments is made up of one of five opsin proteins (LWS, SW1, SWS2, RH2 and RH1) and a chromophore, where each pigment absorbs a different range of light wavelengths. In some non-avian species the spectral sensitivities of these visual pigments have co-evolved with body colour and differ between individuals of the same species. Curiously, however, to date all studies suggest that the visual pigments vary little within the avian class, and even less within a particular species. To investigate further, Ben Knott, a researcher at Deakin University, and Wayne Davies, from the University of Western Australia, both in Australia, and their colleagues turned to the unusual parrot Platycercus elegans whose plumage can range from red to yellow (p. 4454).To begin with, the team characterised the wavelength sensitivity of the different visual pigments, by isolating rod and cone cells expressing the five different pigments. As expected, the five different pigments, absorbed light from the ultraviolet to the red ends of the visual light spectrum.Next, the team moved on to characterising the opsin proteins in more detail and sequenced the mRNA transcripts of the five opsins. As expected, three out of the five (LWS, SWS1 and SWS2) did not vary very much and were similar to opsins found in other bird species. However, the RH1 and RH2 opsins were noticeably different from the equivalent opsins in other birds, with proteins that would have extended carboxyl termini. What's more, the RH2 protein varied considerably, with mRNA transcripts encoding short, medium and long variants. The team suspect that by controlling the size of the RH2 opsin protein, parrots may be able to tweak the spectral sensitivity of the cone cell containing the RH2 visual pigment, thereby aiding them to see better in the yellow and red parts of the spectrum. Although the team still need to test this theory, it seems that there is more variation in avian colour vision than hitherto thought.

Reverse Evolution in RH1 for Adaptation of Cichlids to Water Depth in Lake Tanganyika

Reverse evolution is a widespread phenomenon in biology, but the genetic mechanism for the reversal of a genetic change for adaptation to the ancestral state is not known. Here, we report the first case of complete reverse evolution of two amino acids, serine and alanine, at a single position in RH1 opsin pigment for adaptation to water depth. We determined RH1 sequences of cichlid fishes from four tribes of Lake Tanganyika with different habitat depths. Most of the species were divided into two types: RH1 with 292A for species in shallow water or 292S for species in deep water. Both types were adapted to their ambient light environments as indicated by the absorption spectra of the RH1 pigments. Based on the RH1 locus tree and ecological data, we inferred the ancestral amino acids at position 292 and the distribution of the depth ranges (shallow or deep) of ancestral species of each tribe. According to these estimates, we identified two distinct parallel adaptive evolutions: The replacement A292S occurred at least four times for adaptation from shallow to deep water, and the opposite replacement S292A occurred three times for adaptation from deep to shallow water. The latter parallelism represents the complete reverse evolution from the derived to the ancestral state, following back adaptive mutation with reversal of the RH1 pigment function accompanied by reversal of the species habitat shift.

Development of visual cells in the Pacific bluefin tuna Thunnus orientalis

The development of rod and cone photoreceptor cells was investigated in the retinas of Pacific bluefin tuna larvae and juveniles, using RET-P1 monoclonal antibody labeling to identify photoreceptors. At 60 h after hatching, which was about when feeding began, opsin (presumably green opsin (Rh2)) was expressed in the outer segments of cone cells. At 15 days after hatching (dah), although many labeled cone cells were observed in the dorsal retina, the same type of cone cells had partially appeared in the ventral retina. The presence of rod cell bodies was confirmed by the expression of Rh1 opsin at 15 dah. At 21 dah, the presence of outer segments of rod cells was confirmed by the expression of Rh1 opsin and by morphology. The observations suggest that the cone cells were substantially operable upon the development of their outer segment at around the beginning of the post-larval stage, and the rod cells began to function at around 15 to 21 dah, before and during metamorphosis.

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.

Developmental dynamics of cone photoreceptors in the eel

BackgroundMany fish alter their expressed visual pigments during development. The number of retinal opsins expressed and their type is normally related to the environment in which they live. Eels are known to change the expression of their rod opsins as they mature, but might they also change the expression of their cone opsins?ResultsThe Rh2 and Sws2 opsin sequences from the European Eel were isolated, sequenced and expressed in vitro for an accurate measurement of their λmax values. In situ hybridisation revealed that glass eels express only rh2 opsin in their cone photoreceptors, while larger yellow eels continue to express rh2 opsin in the majority of their cones, but also have <5% of cones which express sws2 opsin. Silver eels showed the same expression pattern as the larger yellow eels. This observation was confirmed by qPCR (quantitative polymerase chain reaction).ConclusionsLarger yellow and silver European eels express two different cone opsins, rh2 and sws2. This work demonstrates that only the Rh2 cone opsin is present in younger fish (smaller yellow and glass), the sws2 opsin being expressed additionally only by older fish and only in <5% of cone cells.

Ontogenetic changes in photoreceptor opsin gene expression in coho salmon(Oncorhynchus kisutch, Walbaum)

Pacific salmonids start life in fresh water then migrate to the sea, after a metamorphic event called smoltification, later returning to their natal freshwater streams to spawn and die. To accommodate changes in visual environments throughout life history, salmon may adjust their spectral sensitivity. We investigated this possibility by examining ontogenetic and thyroid hormone (TH)-induced changes in visual pigments in coho salmon (Oncorhynchus kisutch, Walbaum). Using microspectrophotometry, we measured the spectral absorbance (quantified by lambda(max)) of rods, and middle and long wavelength-sensitive (MWS and LWS) cones in three age classes of coho, representing both freshwater and marine phases. The lambda(max) of MWS and LWS cones differed among freshwater (alevin and parr) and ocean (smolt) phases. The lambda(max) of rods, on the other hand, did not vary, which is evidence that vitamin A(1)/A(2) visual pigment chromophore ratios were similar among freshwater and ocean phases when sampled at the same time of year. Exogenous TH treatment long wavelength shifted the lambda(max) of rods, consistent with an increase in A(2). However, shifts in cones were greater than predicted for a change in chromophore ratio. Real-time quantitative RT-PCR demonstrated that at least two RH2 opsin subtypes were expressed in MWS cones, and these were differentially expressed among alevin, parr and TH-treated alevin groups. Combined with changes in A(1)/A(2) ratio, differential expression of opsin subtypes allows coho to alter the spectral absorbance of their MWS and LWS cones by as much as 60 and 90 nm, respectively. To our knowledge, this is the largest spectral shift reported in a vertebrate photoreceptor.

Effects of exogenous thyroid hormones on visual pigment composition in coho salmon (Oncorhynchus kisutch)

The role of exogenous thyroid hormone on visual pigment content of rod and cone photoreceptors was investigated in coho salmon (Oncorhynchus kisutch). Coho vary the ratio of vitamin A1- and A2-based visual pigments in their eyes. This variability potentially alters spectral sensitivity and thermal stability of the visual pigments. We tested whether the direction of shift in the vitamin A1/A2 ratio, resulting from application of exogenous thyroid hormone, varied in fish of different ages and held under different environmental conditions. Changes in the vitamin A1/A2 visual pigment ratio were estimated by measuring the change in maximum absorbance (lambda max) of rods using microspectrophotometry (MSP). Exogenous thyroid hormone resulted in a long-wavelength shift in rod, middle-wavelength-sensitive (MWS) and long-wavelength-sensitive (LWS) cone photoreceptors. Rod and LWS cone lambda max values increased, consistent with an increase in vitamin A2. MWS cone lambda max values increased more than predicted for a change in the vitamin A1/A2 ratio. To account for this shift, we tested for the expression of multiple RH2 opsin subtypes. We isolated and sequenced a novel RH2 opsin subtype, which had 48 amino acid differences from the previously sequenced coho RH2 opsin. A substitution of glutamate for glutamine at position 122 could partially account for the greater than predicted shift in MWS cone lambda max values. Our findings fit the hypothesis that a variable vitamin A1/A2 ratio provides seasonality in spectral tuning and/or improved thermal stability of visual pigments in the face of seasonal environmental changes, and that multiple RH2 opsin subtypes can provide flexibility in spectral tuning associated with migration-metamorphic events.

Cone visual pigments in two marsupial species: the fat-tailed dunnart (Sminthopsis crassicaudata) and the honey possum (Tarsipes rostratus)

Uniquely for non-primate mammals, three classes of cone photoreceptors have been previously identified by microspectrophotometry in two marsupial species: the polyprotodont fat-tailed dunnart (Sminthopsis crassicaudata) and the diprotodont honey possum (Tarsipes rostratus). This report focuses on the genetic basis for these three pigments. Two cone pigments were amplified from retinal cDNA of both species and identified by phylogenetics as members of the short wavelength-sensitive 1 (SWS1) and long wavelength-sensitive (LWS) opsin classes. In vitro expression of the two sequences from the fat-tailed dunnart confirmed the peak absorbances at 363 nm in the UV for the SWS1 pigment and 533 nm for the LWS pigment. No additional expressed cone opsin sequences that could account for the middle wavelength cones could be amplified. However, amplification from the fat-tailed dunnart genomic DNA with RH1 (rod) opsin primer pairs identified two genes with identical coding regions but sequence differences in introns 2 and 3. Uniquely therefore for a mammal, the fat-tailed dunnart has two copies of an RH1 opsin gene. This raises the possibility that the middle wavelength cones express a rod rather than a cone pigment.

Eel visual pigments revisited: The fate of retinal cones during metamorphosis

During their complex life history, anguilliform eels go through a major metamorphosis when developing from a fresh water yellow eel into a deep-sea silver eel. In addition to major changes in body morphology, the visual system also adapts from a fresh water teleost duplex retina with rods and cones, to a specialized deep-sea retina containing only rods. The history of the rods is well documented with an initial switch from a porphyropsin to a rhodopsin (P523(2) to P501(1)) and then a total change in gene expression with the down regulation of a "freshwater" opsin and its concomitant replacement by the expression of a typical "deep-sea" opsin (P501(1) to P482(1)). Yellow eels possess only two spectral classes of single cones, one sensitive in the green presumably expressing an RH2 opsin gene and the second sensitive in the blue expressing an SWS2 opsin gene. In immature glass eels, entering into rivers from the sea, the cones contain mixtures of rhodopsins and porphyropsins, whereas the fully freshwater yellow eels have cone pigments that are almost pure porphyropsins with peak sensitivities at about 540-545 nm and 435-440 nm, respectively. However, during the early stages of metamorphosis, the pigments switch to rhodopsins with the maximum sensitivity of the "green"-sensitive cone shifting to about 525 nm, somewhat paralleling, but preceding the change in rods. During metamorphosis, the cones are almost completely lost.

Photoreceptor distribution in the retina of adult Pacific salmon: Corner cones express blue opsin

The retina of salmonid fishes has two types of cone photoreceptors: single and double cones. At the nuclear level, these cones are distributed in a square mosaic such that the double cones form the sides of the square and the single cones occupy positions at the centre and at the corners of the square. Double cones consist of two members, one having visual pigment protein maximally sensitive to green light (RH2 opsin), the other maximally sensitive to red light (LWS opsin). Single cones can have opsins maximally sensitive to ultraviolet (UV) or blue light (SWS1 and SWS2 opsins, respectively). In Pacific salmonids, all single cones express UV opsin at hatching. Around the time of yolk sac absorption, single cones start switching opsin expression from UV to blue, in an event that proceeds from the ventral to the dorsal retina. This transformation is accompanied by a loss of single corner cones such that the large juvenile shows corner cones and UV opsin expression in the dorsal retina only. Previous research has shown that adult Pacific salmon have corner cones over large areas of retina suggesting that these cones may be regenerated and that they may express UV opsin. Here we used in-situ hybridization with cRNA probes and RT-PCR to show that: (1) all single cones in non-growth zone areas of the retina express blue opsin and (2) double cone opsin expression alternates around the square mosaic unit. Our results indicate that single cone driven UV sensitivity in adult salmon must emanate from stimulation of growth zone areas.

Heterologous Expression of Bovine Rhodopsin inDrosophilaPhotoreceptor Cells

Vertebrate and invertebrate visual pigments are similar in amino acid sequence, structural organization, spectral properties, and mechanism of action, but possess different chromophores and trigger phototransduction through distinct biochemical pathways. The bovine opsin gene (Rho) was expressed in Drosophila, to examine the properties of a vertebrate opsin within invertebrate photoreceptor cells. Transgenic Drosophila expressing the bovine opsin gene (Rho) in photoreceptors were created. Protein expression and cellular location of bovine rhodopsin was assessed by protein blots and immunofluorescence. The glycosylation state was determined by mobility profiles in SDS-PAGE before and after treatment with endoglycosidase. The rhodopsin chromophore was determined by HPLC-mass spectroscopy (MS) and the spectral properties by spectroscopy. The ability of the bovine rhodopsin to couple to Drosophila phototransduction components was assessed by electroretinography and to couple to vertebrate transducin by light-mediated GTPgammaS-binding assays. Rho showed stable expression even in the absence of endogenous Rh1 opsin and chromophore. It was correctly targeted to the rhabdomeric membranes. Rho remained glycosylated during the maturation process and possessed a distinct glycosylation pattern from that of native Rho. The Drosophila-expressed Rho associated with the 3-hydroxyretinal chromophore but failed to evoke an electroretinogram response from fly photoreceptors. However, the Drosophila-expressed Rho activated transducin in a light-dependent manner. Drosophila photoreceptors express a vertebrate rhodopsin as a functional visual pigment, but the expression does not activate the Drosophila phototransduction pathway. The system allows the characterization and comparison of vertebrate and invertebrate visual pigment properties in a common cell type.