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Non-visual photoreceptors with diverse photopigments allow organisms to adapt to changing light conditions. Whereas visual photoreceptors are involved in image formation, non-visual photoreceptors mainly undertake various non-image-forming tasks. They form specialised photosensory systems that measure the quality and quantity of light and enable appropriate behavioural and physiological responses. Chromatophores are dermal non-visual photoreceptors directly exposed to light and they not only receive ambient photic input but also respond to it. These specialised photosensitive pigment cells enable animals to adjust body coloration to fit environments, and play an important role in mate choice, camouflage and ultraviolet (UV) protection. However, the signalling pathway underlying chromatophore photoresponses and the physiological importance of chromatophore colour change remain under-investigated. Here, we characterised the intrinsic photosensitive system of red chromatophores (erythrophores) in tilapia. Like some non-visual photoreceptors, tilapia erythrophores showed wavelength-dependent photoresponses in two spectral regions: aggregations of inner pigment granules under UV and short-wavelengths and dispersions under middle- and long-wavelengths. The action spectra curve suggested that two primary photopigments exert opposite effects on these light-driven processes: SWS1 (short-wavelength sensitive 1) for aggregations and RH2b (rhodopsin-like) for dispersions. Both western blot and immunohistochemistry showed SWS1 expression in integumentary tissues and erythrophores. The membrane potential of erythrophores depolarised under UV illumination, suggesting that changes in membrane potential are required for photoresponses. These results suggest that SWS1 and RH2b play key roles in mediating intrinsic erythrophore photoresponses in different spectral ranges and this chromatically dependent antagonistic photosensitive mechanism may provide an advantage to detect subtle environmental photic change., (© 2015. Published by The Company of Biologists Ltd.)

Migratory species experience morphological and physiological changes during transitions between different life stages. In particular, modification of sensory systems is critical for animals to adapt to new environments. For example, to prepare for entry into seawater, salmonids undergo smoltification with dramatic changes in ultraviolet photoreceptors and polarized vision, which are important for orientation and foraging behaviours. Extraretinal organs are also involved in photoreception; however, the ontogenetic development of extraretinal photoreceptors is not well known, especially in migratory species. Here, we investigated whether rainbow trout dermal photoreceptors, melanophores, undergo change in spectral sensitivity during smoltification and which candidate molecules may account for this ontogenetic alteration. Our results showed that, contrary to parr melanophores which are insensitive to light, smolt melanophores displayed chromatic photoresponses with the emergence of cryptochrome and melanopsin expression. We suggest that these modifications may benefit the active foraging behaviour of smolts and enable adaptation to variable environments., (© 2014. Published by The Company of Biologists Ltd.)

Non-mammalian vertebrates and invertebrates use extraretinal photoreceptors to detect light and perform diverse non-image-forming functions. Compared to well-studied visual systems, the effect of ambient light conditions on photosensory systems of extraretinal photoreceptors is poorly understood. Chromatophores are photosensitive dermal pigment cells that play an important role in the formation of body color patterns to fit the surrounding environment. Here, we used tilapia erythrophores to investigate the relationship between environmental light and chromatophore photoresponses. All erythrophores from three spectral conditions aggregated their pigment granules in UV/short wavelengths and dispersed in middle/long wavelengths. Unlike retinal visual systems, environmental light did not change the usage of the primary opsins responsible for aggregation and dispersion. In addition, short wavelength-rich and red-shifted background conditions led to an inhibitory effect on erythrophore photoresponses. We suggest that, as extraretinal photoreceptors for non-image-forming functions, chromatophores directly adjust their photoresponse sensitivity via changes in opsin expression levels rather than opsin types when environmental light changes.

Signal reception and production form the basis of animal visual communication, and are largely constrained by environmental light. However, the role of environmental light in producing variation in either signal reception or production has not been fully investigated. To chart the effect of environmental light on visual sensitivity and body colouration throughout ontogeny, we measured spectral sensitivity, lens transmission and body pattern reflectance from juvenile and adult Nile tilapia held under two environmental light treatments. Spectral sensitivity in juveniles reared under a broad-spectrum light treatment and a red-shifted light treatment differed mostly at short wavelengths, where the irradiance of the two light treatments differed the most. In contrast, adults held under the same two light treatments did not differ in spectral sensitivity. Lens transmission in both juveniles and adults did not differ significantly between environmental light treatments, indicating that differences in spectral sensitivity of juveniles originated in the retina. Juveniles and adults held under the two environmental light treatments differed in spectral reflectance, and adults transferred to a third, white light treatment differed in spectral reflectance from their counterparts held under the two original treatments. These results demonstrate that environmental light plays a crucial role in shaping signal reception in juveniles and signal production throughout ontogeny, reinforcing the notion that environmental light has the capacity to influence animal communication, and suggesting that the characteristics of environmental light should be considered in models of ecological speciation.

Dermal specialized pigment cells (chromatophores) are thought to be one type of extraretinal photoreceptors responsible for a wide variety of sensory tasks, including adjusting body coloration. Unlike the well-studied image-forming function in retinal photoreceptors, direct evidence characterizing the mechanism of chromatophore photoresponses is less understood, particularly at the molecular and cellular levels. In the present study, cone opsin expression was detected in tilapia caudal fin where photosensitive chromatophores exist. Single-cell RT-PCR revealed co-existence of different cone opsins within melanophores and erythrophores. By stimulating cells with six wavelengths ranging from 380 to 580 nm, we found melanophores and erythrophores showed distinct photoresponses. After exposed to light, regardless of wavelength presentation, melanophores dispersed and maintained cell shape in an expansion stage by shuttling pigment granules. Conversely, erythrophores aggregated or dispersed pigment granules when exposed to short- or middle/long-wavelength light, respectively. These results suggest that diverse molecular mechanisms and light-detecting strategies may be employed by different types of tilapia chromatophores, which are instrumental in pigment pattern formation.

Background: Two competing theories have been advanced to explain the evolution of multiple cone classes in vertebrate eyes. These two theories have important, but different, implications for our understanding of the design and tuning of vertebrate visual systems. The 'contrast theory' proposes that multiple cone classes evolved in shallow-water fish to maximize the visual contrast of objects against diverse backgrounds. The competing 'flicker theory' states that multiple cone classes evolved to eliminate the light flicker inherent in shallow-water environments through antagonistic neural interactions, thereby enhancing object detection. However, the selective pressures that have driven the evolution of multiple cone classes remain largely obscure., Results: We show that two critical assumptions of the flicker theory are violated. We found that the amplitude and temporal frequency of flicker vary over the visible spectrum, precluding its cancellation by simple antagonistic interactions between the output signals of cones. Moreover, we found that the temporal frequency of flicker matches the frequency where sensitivity is maximal in a wide range of fish taxa, suggesting that the flicker may actually enhance the detection of objects. Finally, using modeling of the chromatic contrast between fish pattern and background under flickering illumination, we found that the spectral sensitivity of cones in a cichlid focal species is optimally tuned to maximize the visual contrast between fish pattern and background, instead of to produce a flicker-free visual signal., Conclusions: The violation of its two critical assumptions substantially undermines support for the flicker theory as originally formulated. While this alone does not support the contrast theory, comparison of the contrast and flicker theories revealed that the visual system of our focal species was tuned as predicted by the contrast theory rather than by the flicker theory (or by some combination of the two). Thus, these findings challenge key assumptions of the flicker theory, leaving the contrast theory as the most parsimonious and tenable account of the evolution of multiple cone classes.

Color vision is most beneficial when the visual system is color constant and can correct the excitations of photoreceptors for differences in environmental irradiance. A phenomenon related to color constancy is color induction, where the color of an object shifts away from the color of its surroundings. These two phenomena depend on chromatic spatial integration, which was suggested to originate at the feedback synapse from horizontal cells (HC) to cones. However, the exact retinal site was never determined. Using the electroretinogram and compound action potential recordings, we estimated the spectral sensitivity of the photoresponse of cones, the output of cones, and the optic nerve in rainbow trout. Recordings were performed before and following pharmacological inhibition of HC-cone feedback, and were repeated under two colored backgrounds to estimate the efficiency of color induction. No color induction could be detected in the photoresponse of cones. However, the efficiency of color induction in the cone output and optic nerve was substantial, with the efficiency in the optic nerve being significantly higher than in the cone output. We found that the efficiency of color induction in the cone output and optic nerve decreased significantly with the inhibition of HC-cone feedback. Therefore, our findings suggest not only that color induction originates as a result of HC-cone feedback, but also that this effect of HC-cone feedback is further amplified at downstream retinal elements, possibly through feedback mechanisms at the inner plexiform layer. This study provides evidence for an important role of HC-cone feedback in mediating color induction, and therefore, likely also in mediating color constancy.

Humans use three cone photoreceptor classes for colour vision, yet many birds, reptiles and shallow-water fish are tetrachromatic and use four cone classes. Screening pigments, which narrow the spectrum of photoreceptors in birds and diurnal reptiles, render visual systems with four cone classes more efficient. To date, however, the question of tetrachromacy in shallow-water fish that, like humans, lack screening pigments, is still unsolved. We raise the possibility that tetrachromacy in fish has evolved in response to higher spectral complexity of underwater light. We compared the dimensionality of colour vision in humans and fish by examining the spectral complexity of the colour signal reflected from objects into their eyes. We show that fish require four to six cone classes to reconstruct the colour signal of aquatic objects at the accuracy level achieved by humans viewing terrestrial objects. This is because environmental light, which alters the colour signals, is more complex and contains more spectral fluctuations underwater than on land. We further show that fish cones are better suited than human cones to detect these spectral fluctuations, suggesting that the capability of fish cones to detect high-frequency fluctuations in the colour signal confers an advantage. Taken together, we propose that tetrachromacy in fish has evolved to enhance the reconstruction of complex colour signals in shallow aquatic environments. Of course, shallow-water fish might possess fewer than four cone classes; however, this would come with the inevitable loss in accuracy of signal reconstruction.

Polarization sensitivity (PS) in vertebrate vision is controversial, perhaps because its underlying mechanism has remained obscure. An issue that might have added to the controversy is that rainbow trout (Oncorhynchus mykiss), which have served as the primary model system for polarization-based orientation, lose their ability to orient relative to celestial polarized-light patterns when parr (fry) transform into migratory smolts (juveniles), which would benefit most from polarization-based orientation. Here we addressed two key questions: (1) what is the mechanism underling PS?, and (2) how can the paradoxical loss of PS in trout smolts be reconciled? We assessed PS from optic nerve recordings in parr and smolts and found that the retinal region with enhanced PS shifted from the ventral retina in parr to the dorsal retina in smolts. This adaptation may allow fish to use the most reliable polarization field encountered at each life stage, the celestial polarization field in the shallow-swimming parr and the depth-insensitive underwater polarization field in the deep-swimming smolts. In addition, we assessed spectral sensitivity across the retina and during ontogeny and fit a cascade retinal model to PS data. We found that differential contribution of two cone detectors with orthogonal PS could drive the variation in PS and that feedback from horizontal cells to cones could explain the differential amplification of PS. This elegant arrangement, in which weak PS of cones is amplified and tuned by retinal networks, allows for PS without interfering with sampling of other visual information and illustrates how sensory systems may simultaneously process disparate aspects of physical environments.

Retinal neurogenesis in fish facilitates cellular rearrangement throughout ontogeny, potentially allowing for optimization of the visual system to shifts in habitat and behaviour. To test this possibility, we studied the developmental trajectory of the photopic visual process in the Nile tilapia. We examined ontogenetic changes in lens transmission, photoreceptor sensitivity and post-receptoral sensitivity, and used these to estimate changes in cone pigment frequency and retinal circuitry. We observed an ontogenetic decrease in ultraviolet (UV) photoreceptor sensitivity, which resulted from a reduction in the SWS1 cone pigment frequency, and was associated with a reduction in lens transmission at UV wavelengths. Additionally, post-receptoral sensitivity to both UV and long wavelengths decreased with age, probably reflecting changes in photoreceptor sensitivity and retinal circuitry. This novel remodelling of retinal circuitry occurred following maturation of the visual system but prior to reaching adulthood, and thus may facilitate optimization of the visual system to the changing sensory demands. Interestingly, the changes in post-receptoral sensitivity to long wavelengths could not be predicted by the changes observed in lens transmission, cone pigment frequency or photoreceptor sensitivity. This finding emphasizes the importance of considering knowledge of visual sensitivity and retinal processing when studying visual adaptations and attempting to relate visual function to the natural environment. This study advances our understanding of ontogeny in visual systems and demonstrates that the association between different elements of the visual process can be explored effectively by examining visual function throughout ontogeny.

Thyroid hormones (THs) play a vital role in vertebrate neural development, and, together with the beta isoform of the thyroid hormone receptor (TRβ), the development and differentiation of cone photoreceptors in the vertebrate retina. Rainbow trout undergo a natural process of cone cell degeneration during development and this change in photoreceptor distribution is regulated by thyroxine (T4; a thyroid hormone). In an effort to further understand the role of T4 in photoreceptor ontogeny and later developmental changes in photoreceptor subtype distribution, the influence of enhanced in ovo T4 content on the onset of opsin expression and cone development was examined. Juvenile trout reared from the initial in ovo-treated embryos were challenged with exogenous T4 at the parr stage of development to determine if altered embryonic exposure to yolk THs would affect later T4-induced short-wavelength-sensitive (SWS1) opsin transcript downregulation and ultraviolet-sensitive (UVS) cone loss. In ovo TH manipulation led to upregulation of both SWS1 and long-wavelength-sensitive (LWS) opsin transcripts in the pre-hatch rainbow trout retina and to changes in the relative expression of TRβ. After 7 days of exposure to T4, juveniles that were also treated with T4 in ovo had greatly reduced SWS1 expression levels and premature loss of UVS cones relative to T4-treated juveniles raised from untreated eggs. These results suggest that changes in egg TH levels can have significant consequences much later in development, particularly in the retina.

Lake Malawi boasts the highest diversity of freshwater fishes in the world. Nearshore sites are categorized according to their bottom substrate, rock or sand, and these habitats host divergent assemblages of cichlid fishes. Sexual selection driven by mate choice in cichlids led to spectacular diversification in male nuptial coloration. This suggests that the spectral radiance contrast of fish, the main determinant of visibility under water, plays a crucial role in cichlid visual communication. This study provides the first detailed description of underwater irradiance, radiance and beam attenuation at selected sites representing two major habitats in Lake Malawi. These quantities are essential for estimating radiance contrast and, thus, the constraints imposed on fish body coloration. Irradiance spectra in the sand habitat were shifted to longer wavelengths compared with those in the rock habitat. Beam attenuation in the sand habitat was higher than in the rock habitat. The effects of water depth, bottom depth and proximity to the lake bottom on radiometric quantities are discussed. The radiance contrast of targets exhibiting diffused and spectrally uniform reflectance depended on habitat type in deep water but not in shallow water. In deep water, radiance contrast of such targets was maximal at long wavelengths in the sand habitat and at short wavelengths in the rock habitat. Thus, to achieve conspicuousness, color patterns of rock- and sand-dwelling cichlids would be restricted to short and long wavelengths, respectively. This study provides a useful platform for the examination of cichlid visual communication.

Background: Color vision plays a critical role in visual behavior. An animal's capacity for color vision rests on the presence of differentially sensitive cone photoreceptors. Spectral sensitivity is a measure of the visual responsiveness of these cones at different light wavelengths. Four classes of cone pigments have been identified in vertebrates, but in teleost fishes, opsin genes have undergone gene duplication events and thus can produce a larger number of spectrally distinct cone pigments. In this study, we examine the question of large-scale variation in color vision with respect to individual, sex and species that may result from differential expression of cone pigments. Cichlid fishes are an excellent model system for examining variation in spectral sensitivity because they have seven distinct cone opsin genes that are differentially expressed., Results: To examine the variation in the number of cones that participate in cichlid spectral sensitivity, we used whole organism electrophysiology, opsin gene expression and empirical modeling. Examination of over 100 spectral sensitivity curves from 34 individuals of three species revealed that (1) spectral sensitivity of individual cichlids was based on different subsets of four or five cone pigments, (2) spectral sensitivity was shaped by multiple cone interactions and (3) spectral sensitivity differed between species and correlated with foraging mode and the spectral reflectance of conspecifics. Our data also suggest that there may be significant differences in opsin gene expression between the sexes., Conclusions: Our study describes complex opponent and nonopponent cone interactions that represent the requisite neural processing for color vision. We present the first comprehensive evidence for pentachromatic color vision in vertebrates, which offers the potential for extraordinary spectral discrimination capabilities. We show that opsin gene expression in cichlids, and possibly also spectral sensitivity, may be sex-dependent. We argue that females and males sample their visual environment differently, providing a neural basis for sexually dimorphic visual behaviour. The diversification of spectral sensitivity likely contributes to sensory adaptations that enhance the contrast of transparent prey and the detection of optical signals from conspecifics, suggesting a role for both natural and sexual selection in tuning color vision.

The UVS cone mechanism is known to light adapt at low intensities in comparison to other cones. We were interested in whether this property was related to higher sensitivity in UVS cones or to network adjustments in sensitivity. We investigated spectral sensitivity of 107 individual cone photoreceptors in rainbow trout (Oncorhynchus mykiss) using a whole-cell voltage clamp technique. Mean time-to-peak response was 339+/-90ms and flash sensitivity for a 100ms flash was 4.37x10(-3)+/-2.50x10(-3)pAphotons(-1)microm(2), with no significant differences between the UVS, SWS, MWS and LWS cone classes. The spectral sensitivity of the UVS, SWS and LWS cones conformed to the expression of SWS1, SWS2 and LWS opsin genes. The spectral sensitivity of MWS cones, however, showed clear evidence of co-expression of RH2a and RH2b opsin pigments. The fish used in this study bridged the ontogenetic stage where the MWS cones shift their expression from RH2b to RH2a., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We were interested in comparing the characteristics of polarization sensitivity in Atlantic salmon to those in Pacific salmon. Here we show that the common ancestor to the clade containing Salmo salar, Oncorhynchus mykiss, O. nerka, O. clarkii and Salvelinus fontinalis has the trait of ultraviolet polarization sensitivity. We examined spectral and polarization sensitivity of juvenile Atlantic salmon (Salmo salar) using both optic nerve compound action potential (CAP) and electroretinogram (ERG) recordings. Our experiments employed photic manipulation to adjust the sensitivity of the four cone mechanisms of Atlantic salmon. A spectrally broad background was used to ensure a contribution of all cone mechanisms to both spectral and polarization sensitivity. Chromatic adaptation was used to isolate the sensitivity of each of the four cone mechanisms for both spectral and polarization sensitivity. Under spectrally broad conditions, UV sensitive (UVS), mid wavelength sensitive (MWS) and long wavelength sensitive (LWS) cone mechanisms contributed to polarization sensitivity. CAP recordings produced the typical 'W' shaped polarization sensitivity curve reflecting two active polarization detectors with peaks at e-vector orientations of 0 deg, 90 deg and 180 deg, and troughs at 30 deg and 150 deg. ERG recordings produced a four-peaked polarization sensitivity curve reflecting two active polarization detectors and negative feedback activity, with peaks at e-vectors 0 deg, 45 deg, 90 deg, 135 deg and 180 deg, and troughs at 30 deg, 60 deg, 120 deg and 150 deg. Polarization-sensitivity measurements of isolated cone mechanisms revealed two orthogonal polarization detector mechanisms in Atlantic salmon, identical to that found in rainbow trout and other Pacific salmonid fishes. Moreover, under spectrally broad background conditions, CAP and ERG polarization sensitivity of Atlantic salmon did not differ significantly from that reported in Pacific salmonids.

The cichlid fish radiations of the African Great Lakes are an important model for evolutionary biology. Cichlids have diverse colour vision systems and predominately express three cone visual pigments. However, rare populations of spectrally distinct cones have been found in a number of species, but it is not known whether they contribute to spectral sensitivity. Adult Nile tilapia, Oreochromis niloticus, an ancestral outgroup to the cichlid radiations in the Great Lakes, have three cone types: short-wavelength sensitive (SWS), medium-wavelength sensitive (MWS) and long-wavelength sensitive (LWS) cones, but evidence from microspectrophotometry and cone opsin gene expression suggests they may also have violet-sensitive (VS) cones. We used electrophysiology to assess spectral sensitivity in this species and found evidence of four sensitivity peaks in the ranges 380-420, 440-480, 500-600 and 600-680 nm, with maximal sensitivity at longer wavelengths. The continued presence of a 380-420 nm peak under long-wavelength chromatic adapting backgrounds indicates that this is due to a VS cone mechanism not the beta-band of the LWS cone mechanism. Differences in spectral sensitivity curves recorded at different times of year revealed evidence of A1/A2 shifts. The presence of notches in the sensitivity curves and a multiple-mechanisms model used to assess cone contributions indicated that the curves are the result of four cone mechanisms (VS, SWS, MWS and LWS cones) and that chromatically opponent processes occur between mechanisms. The spectral transmittance of the lens steeply declines between 410-380 nm, limiting the short-wavelength limb of the VS cone. As adults, Nile tilapia appear to possess the necessary retinal mechanisms for colour vision. While maximal sensitivity to longer wavelengths is an adaptation to the wavelengths of light predominantly available in their natural habitats, their broad sensitivity range suggests that Nile tilapia possess a flexible, generalised visual system able to adapt to changes in visual environment in their highly variable natural habitat.

Rainbow trout possess ultraviolet-sensitive (UVS) cones in their retina that degenerate naturally during development. This phenomenon can be induced with exogenous thyroxine [T4, a thyroid hormone (TH)] treatment. However, the previous T4 exposure experiments employed static water immersion; a method that could introduce confounding stress effects on the fish. Because of this, it was uncertain if T4 alone was sufficient to induce retinal changes or if stress-related hormones were also necessary to initiate this process. Furthermore, it was unclear whether endogenous T4 was the factor responsible for initiating natural UVS cone loss during development. The current study examined the role of systemic T4 on the juvenile rainbow trout retina using a slow-release implant. Exogenous T4 treatment resulted in SWS1 opsin downregulation and UVS cone loss after four weeks of exposure, signifying that T4 is sufficient to induce this process. Blocking endogenous T4 production with propylthiouracil (PTU, an anti-thyroid agent) attenuated SWS1 downregulation and UVS cone loss in the retina of naturally developing rainbow trout, suggesting that endogenous T4 is necessary to initiate retinal remodelling during development. Quantitative real-time RT-PCR analysis demonstrated that several TH-regulating components are expressed in the trout retina, and that expression levels of the TH receptor isoform TRbeta and the type 2 deiodinase (D2) change with T4 treatment. This suggests that T4 may act directly on the retina to induce UVS cone loss. Taken together, these results demonstrate that systemic TH is necessary and sufficient to induce SWS1 opsin downregulation and UVS cone loss in the retina of juvenile rainbow trout.

Rainbow trout undergo natural cone degeneration and thus are interesting models for examining mechanisms of neural degeneration. They have ultraviolet-sensitive (UVS) cones that are lost over most of the retina during development; only a small functional population remains in the dorsal retina. How this spatial distribution of UVS cones is maintained is unclear. Thyroxine (T4) induces UVS cone loss, and local thyroid hormone regulation was hypothesized to control UVS cone distribution. Thyroid hormone receptor alpha (TRalpha), thyroid hormone receptor beta (TRbeta) and Type 2 deiodinase (D2) regulate thyroid hormone exposure to target cells. Regional retinal expression of these genes was investigated during exogenous T4 treatment and natural smoltification of rainbow trout. Each retina from dark-adapted parr, T4-treated parr and natural smolts was divided into four quadrants, and total RNA was isolated. Quantitative real-time RT-PCR analysis demonstrated that all retinal quadrants had increased accumulation of TRbeta transcripts 2 days post-T4 treatment, corresponding to initiation of SWS1 opsin downregulation. Smolts exhibited decreased accumulation of TRalpha and TRbeta transcripts in all quadrants, but this effect was most pronounced in the dorso-temporal (DT) retinal quadrant where UVS cones persist. By contrast, in 2 day T4-treated parr, the DT quadrant showed increased expression of TRalpha and TRbeta. Furthermore, D2 transcripts decreased in the DT quadrant of T4-treated parr but increased in the DT quadrant of smolts. These results suggest that T4 upregulates TRbeta expression to initiate SWS1 opsin downregulation, while TRalpha and TRbeta downregulation occurs to prevent natural loss of UVS cones from the DT retina.

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.

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.

A number of teleost fishes have photoreceptor mechanisms to detect linearly polarized light. We studied the neuronal mechanism underlying this ability. It was found that a polarized signal could be detected in rainbow trout (Oncorhynchus mykiss) both in the electroretinogram (ERG) and in the compound action potential (CAP) measured in the optic nerve, indicating a strong retinal contribution to the processing of polarized light. The CAP recordings showed a W-shaped sensitivity curve, with a peak at 0 degrees , 90 degrees and 180 degrees , consistent with processes for both vertical and horizontal orientation. By contrast, the ERG recordings reveal a more complex pattern. In addition to the peaks at 0 degrees , 90 degrees and 180 degrees , two additional peaks appeared at 45 degrees and 135 degrees . This result suggests a specialized contribution of the outer retina in the processing of polarized light. The spectral sensitivity of the mechanisms responsible for these intermediate peaks was studied using chromatic adaptation. Here we show that long wavelength-sensitive (LWS) cone mechanism adaptation shifted the intermediate peaks towards 90 degrees , whereas ultraviolet-sensitive (UVS) cone mechanism adaptation shifted the peaks away from 90 degrees towards either 0 degrees or 180 degrees . These results provide further confirmation that the 90 degrees peak is dominated by the LWS cone mechanism and the 0 degrees and 180 degrees peaks are dominated by the UVS cone mechanism. In addition, a pharmacological approach was used to examine the retinal neural mechanisms underlying polarization sensitivity. The effect of blocking negative feedback from horizontal cells to cones on the ERG was studied by making intraocular injections of low doses of cobalt, known to block this feedback pathway. It was found that the intermediate peaks seen in the ERG polarization sensitivity curves were eliminated after application of cobalt, suggesting that these peaks are due to outer retinal inhibition derived from feedback of horizontal cells onto cones. A simple computational model was developed to evaluate these results. The model consists of opponent and non-opponent processing elements for the two polarization detectors. This model provides a first approximation analysis suggesting that opponent processing occurs in the outer retina for polarization vision. Although it seems that polarization vision uses a slightly more complicated coding scheme than colour vision, the results presented in this paper suggest that opponent and non-opponent channels process polarization information.

Purpose: Analyses that reveal the relative abundance of proteins are informative in elucidating mechanisms of retinal development and disease progression. However, popular high-throughput proteomic methods do not reliably detect opsin protein abundance, which serve as markers of photoreceptor differentiation. We utilized thyroid-hormone (TH) treatment of rainbow trout (Oncorhynchus mykiss) as a model of cone apoptosis and cone regeneration. We used this model to investigate if emerging proteomic technology allows effective analysis of retinal development and opsin protein abundance. We also sought to begin a characterization of proteomic changes in the retina occurring with TH treatment and address whether TH affects proliferation or photoreceptor differentiation., Methods: Retinal homogenates were prepared from control and TH-treated fish. Peptides from control and treated homogenates were differentially labeled, using isotope-code affinity tags (ICAT) and analyzed using capillary liquid chromatography-electrospray ionization-tandem mass spectrometry (capLC-ESI-MS/MS). This method identifies proteins and quantifies their relative abundance between two samples., Results: The relative abundance of many retinal proteins changed during TH treatment. These included proteins from every functional class. We detected 1,684 different peptides, and our quantification suggests that 94 increased and 146 decreased in abundance more than 50% during TH treatment. Cell-cycle proteins appear to be increased, consistent with TH-inducing cell proliferation, similar to its effect in Xenopus. Other proteins associated with retinal development, such as deltaA and tubulins, changed in abundance during TH treatment. Rod opsin and three cone opsins were identified and the relative abundance of each changed with TH treatment., Conclusions: ICAT and capLC-ESI-MS/MS are an effective complement to other molecular approaches that investigate the mechanisms of retinal development. Unlike other proteomic techniques, this approach does not require development of species- or tissue-specific methodology, such as characterizing two dimensional (2D) gels or antibodies, in order to be practical as a high-throughput approach. Importantly, this technology was able to assess the relative abundance of opsin proteins. These findings represent the first high-throughput proteomic analysis of the retina and demonstrate the technique's ability to provide useful information in retinal development.

In this study, we demonstrate the capacity for damselfish (green chromis, Chromis viridis) to discriminate between different e-vector orientations of ultraviolet polarized light. We examined the ability of green chromis to resolve small differences in e-vector orientation of ultraviolet polarized light. Fish were successfully trained to swim towards an e-vector orientation of polarized light using a behavioural chamber. C. viridis was able to discriminate between the horizontal and the vertical plane of ultraviolet polarized light independent of brightness content of the stimuli. However, e-vector discrimination capability disappeared when the ultraviolet portion of the light stimuli was removed, indicating that the presence of ultraviolet light was critical for e-vector discrimination. Fish could also distinguish between relatively small e-vector orientations of ultraviolet polarized light. Functional implications for high e-vector discriminative capabilities could be used in functional domains such as feeding and communication.

Using a behavioral assay based on visually mediated escape responses, we measured long-wavelength-sensitive red cone (LC) sensitivities in zebrafish. In a 24 h period, the zebrafish were least sensitive to red light in the early morning and most sensitive in the late afternoon. To investigate if the fluctuation of behavioral cone sensitivity correlates with opsin gene expression, we measured LC opsin mRNA expression at different times in the day and night under different lighting conditions. Under a normal light-dark cycle, the expression of LC opsin mRNA determined by real-time RT-PCR was low in the early morning and high in the late afternoon, similar to the fluctuation of behavioral cone sensitivity. This rhythm of LC opsin mRNA expression, however, dampened out gradually in constant conditions. After 24 h of constant light (LL), the expression of LC opsin mRNA dropped to levels similar to those determined in the early morning in control animals. By contrast, when the zebrafish were kept in constant dark (DD), the expression of LC opsin mRNA increased, to levels about 30-fold higher than the expression in the early morning in control animals. This day-night fluctuation in LC opsin mRNA expression was correlated to changes in opsin density in the outer segment of cone photoreceptor cells. Microspectrophotometry (MSP) measurements found significant differences in red cone outer segment optical density with a rhythm following the behavioral sensitivity. Furthermore, dopamine modulated the circadian rhythms in expression of LC opsin mRNA. Administration of dopamine increased LC opsin mRNA expression, but only in the early morning.

Positive selection can be demonstrated by statistical analysis when non-synonymous nucleotide substitutions occur more frequently than synonymous substitutions (dN>dS). This pattern of sequence evolution has been observed in the rhodopsin gene of cichlids. Mutations in opsin genes resulting in amino acid (AA) replacement appear to be associated with the evolution of specific color patterns and the evolution of courtship behaviors. Within fish, AA replacements in opsin proteins have improved vision at great depths and have occurred in deep-sea species. Salmonids experience diverse photic environments during their life history. Furthermore, sexual selection has resulted in species-specific male and female coloration during spawning. To look for evidence of positive selection in salmonid opsins, we sequenced the RH1, RH2, LWS, SWS1, and SWS2 genes from six Pacific salmon species as well as the Atlantic salmon. These salmonids include landlocked and migratory species and species that vary in their coloration during spawning. In each opsin gene comparison from all species sampled, traditional dN:dS analysis did not indicate positive selection. However, the more sensitive Creevey-McInerney statistical analysis indicates that RH1 and RH2 experienced positive selection early in the evolution and speciation of salmonids.

Previous studies in our laboratory have examined the loss of ultraviolet-sensitive (UVS) cones and UV sensitivity. This study looks at the question of regeneration of UVS cones and its topographic distribution, along with several other measures of the cone mosaic. Topography of the cone mosaic in rainbow trout smolts (post-metamorphic juveniles) was examined under normal growth conditions and during an exogenous thyroid hormone (TH) challenge. Growth of trout retina was studied over six weeks. Retinas sampled at 0, 3 and 6 weeks were embedded in EPON resin, and thick (1 micro m) tangential sections were stained with Richardson's stain. Sites representing central ventral, ventral, temporal, dorsal and nasal retina were sampled. Variables measured were cone densities, mean double cone diameter and mean spacing between cones of the same type. These same variables were compared with those of fish that were challenged with L-thyroxin (T4), and regeneration of UVS cones was assessed. Principal components of the correlation matrix of all photoreceptor measurements were analysed using analysis of variance. Here, we show several interesting effects of thyroxin exposure on post-metamorphic rainbow trout: (1) controls at week 0 have a high density of UVS cones in the temporal and dorsal sampling regions and a high density of blue (short-wavelength)-sensitive (SWS) and double cones across all regions sampled; (2) both control and TH-treated fish had less abundant, larger and less tightly packed SWS and double cones and a lower density of UVS cones in the temporal and dorsal sampling regions three and six weeks into the experiment compared with the starting condition at week 0; (3) fish treated with TH had a higher UVS cone density in the nasal and ventral sampling regions and there were higher densities of SWS and double cones in the central ventral, temporal and ventral regions, but lower densities in the nasal sampling regions, relative to the controls. The regeneration of UVS cones into the ventral retinal hemisphere in post-juvenile salmonids has important implications for visually guided behavior.

We have developed a charge-coupled-device (CCD)-based microspectrophotometer (MSP) system and provide the first report on the successful employment of this technology to measure the spectral absorbance properties of vertebrate photoreceptors. The principal difference between the CCD-based MSP system and wavelength-scanning MSP systems, commonly used in vision biology, is that a short duration (800-1200 ms), broad-spectrum flash is employed rather than ascending and descending wavelength scanning. Data acquisition is thus significantly faster, with the added possible advantages of less variance due to movement of target photoreceptors during measurement, reduced spectral distortion due to photoproduct interference and an ability to measure fast, transient changes in absorbance as bleaching proceeds. Rainbow trout photoreceptors, previously measured with a wavelength-scanning MSP system, were again measured using the CCD-based MSP system. Our analysis of optical recordings from 102 photoreceptors corroborated data obtained previously with rainbow trout photoreceptors on lambda(max) (wavelength of maximum absorbance), A(max) (maximum absorbance) and half maximum bandwidth (HBW) of ultraviolet-, blue-, green- and red-sensitive cones and rods. There were slight differences in lambda(max) and half-maximum bandwidth of the ultraviolet-, blue- and green-sensitive cone classes, but this was most probably due to variation in the A(1):A(2) visual pigment ratio of the trout used in the two different studies. However, we were capable of resolving the A(1) and A(2) visual pigment spectra in the red-sensitive cones and the rods.

Ultraviolet visual sensitivity appears to be reduced and, possibly, lost during smoltification in anadromous populations of salmonid fishes. Similar changes occur in non-anadromous salmonids over a mass range that is associated with smoltification in their anadromous conspecifics. However, in sexually mature adult salmonids, ultraviolet-sensitive cones are present in the dorso-temporal retina, suggesting that ultraviolet sensitivity (i) may be regained with sexual maturity or (ii) might never be completely lost. Both smoltification and the transition to sexual maturity are regulated, in part, by the hormone thyroxine. Thyroxine treatment of juvenile Oncorhynchus mykiss results in precocial developmental changes that mimic smoltification, including a reduction of ultraviolet sensitivity. However, whether loss of ultraviolet sensitivity in O. mykiss or in other species of salmonids is complete during normal development (or in response to thyroxine treatment) is unclear. In the present study, we have 'mapped' topographically ultraviolet photosensitivity during natural and hormone-induced smoltification. Thyroxine-treated O. mykiss juveniles and anadromous steelhead O. mykiss smolts were examined for ultraviolet visual sensitivity by recording compound action potentials from the optic nerve. By selectively illuminating either the dorsal or the ventral retina, we have shown that the reduction of ultraviolet sensitivity occurs primarily in the ventral retina in both groups of fish. Ultraviolet sensitivity remains intact in the dorsal retina.

Polarization vision in vertebrates has been marked with significant controversy over recent decades. In the last decade, however, models from two laboratories have indicated that the spatial arrangement of photoreceptors provides the basis for polarization sensitivity Work in my laboratory, in collaboration with I. Novales Flamarique and F. I. Harosi, has shown that polarization sensitivity depends on a well-defined square cone mosaic pattern and that the biophysical properties of the square cone mosaic probably account for polarization vision in the ultraviolet spectrum. The biophysical mechanism appears to be based on the selective reflection of axial-polarized light by the partitioning membrane, formed along the contact zone between the members of the double cones, onto neighbouring ultraviolet-sensitive cones. In this short review, I discuss the historical development of this research problem.

Spectral and polarisation sensitivity were compared among juvenile (parr) rainbow trout (Oncorhynchus mykiss), steelhead (O. mykiss), cutthroat trout (O. clarki clarki), kokanee (O. nerka) and brook char (Salvelinus fontinalis) using multi-unit recording from the optic nerve. Although reared under the same conditions, differences in photopic spectral sensitivity were evident. Specifically, ON-responses were co-dominated by L- and M-cone mechanisms in all fish except O. nerka, consistent with an M-cone mechanism sensitivity. The sensitivity of OFF-responses was dominated by the M-cone mechanism for all fish, but O. mykiss appeared to show an additional contribution from the L-cone mechanism. Using chromatic adaptation, an independent ultraviolet-sensitive mechanism is described for the first time for the salmonid genus Salvelinus. In addition, this ultraviolet-cone mechanism was present in the members of the genus Oncorhynchus that were examined. Thus, ultraviolet sensitivity appears to be common to the major extant clades of the subfamily Salmoninae. All species showed differential sensitivity to both vertical and horizontal linearly polarised light. This sensitivity differed between ON- and OFF-responses. The ON-responses were maximally sensitive to both vertically and horizontally polarised light, whereas the OFF-responses displayed maximal sensitivity to horizontally polarised light in all species, with reduced sensitivity to vertically polarised light compared with ON-responses. Because of the similarity in the physiological characteristics of polarisation sensitivity among the salmonid species examined, no relationship between the degree of migratory tendency and the ability to detect polarised light could be identified.

Although color-opponent neurons appear to subserve color vision, precisely how these cells encode hue is still not clear. Single-unit, extracellular recordings from the rainbow trout optic tectum were made in order to examine the possible role of action potential timing in coding chromatic stimuli. We found that color-opponent units can exhibit differences in response latency which are a function of wavelength and response sign, with the OFF response exhibiting the shorter response latency. We also found that units often responded with spike bursts characterized by early and late spikes separated by a silent period, with the relative proportion of early and late spikes varying as a function of wavelength. This type of discharge pattern appears to be a result of inhibitory, color-opponent processes. We suggest that complete inhibition of early spikes may be the mechanism underlying the observed latency differences. These findings suggest a role for action potential patterning in coding chromatic stimuli.

Juvenile rainbow trout (Oncorhynchus mykiss) were given ethambutol (900 mg kg-1 body mass per day) for 4 weeks to examine its effects on vision. Using multi-unit recording from the optic nerve, spectral sensitivity of the on-responses were significantly affected in two regions, 340-440 nm and 600-660 nm. Off-responses were statistically unaffected. Changes in sensitivity to polarised light were also observed with on-responses to vertically-polarized light decreasing relative to horizontally-polarised light. In contrast, off-responses were less affected. The treatment effects were attributed to changes in the relative contribution of the photoreceptor channels as recorded at the level of the optic nerve.

The sensitivities of two species of sunfish (Lepomis gibbosus and Lepomis cyanellus) to the electric field (E-vector) of polarized light were assessed by compound action potential recordings from the optic nerve of live fish. Under white light and long wavelength adapting backgrounds, two cone mechanisms were found with maximum sensitivities in the long wavelength (lambda max approximately 620 nm) and middle wavelength (lambda max approximately 530 nm) regions of the spectrum. In contrast to previous findings (Cameron & Pugh, 1991), no evidence of polarization sensitivity was observed for either species. We conclude from these results that post-larval sunfish do not exhibit polarization sensitivity.

We measured the spectral distributions of the underwater total and polarized light fields in the upper photic zone of meso-eutrophic waters (i.e., blue-green waters containing medium to high chlorophyll a concentrations). Per cent polarization levels during the day were always lower than 40%, but at crepuscular times these values could increase to 67%. A corresponding change occurred in the spectral distribution, with proportionately more shorter wavelength photons contributing to the total spectrum during crepuscular periods. Electrophysiological recordings from the optic nerve of rainbow trout subjected to light stimuli of varying polarization percentages show that the animal's threshold for detecting polarized light is between 63 and 72%. These physiological findings suggest that the use of water-induced polarized light cues by rainbow trout and similar percomorph fish should be restricted to crepuscular time periods.