Long-wavelength-sensitive Research Articles

S cones: Evolution, retinal distribution, development, and spectral sensitivity

S cones expressing the short wavelength-sensitive type 1 (SWS1) class of visual pigment generally form only a minority type of cone photoreceptor within the vertebrate duplex retina. Hence, their primary role is in color vision, not in high acuity vision. In mammals, S cones may be present as a constant fraction of the cones across the retina, may be restricted to certain regions of the retina or may form a gradient across the retina, and in some species, there is coexpression of SWS1 and the long wavelength-sensitive (LWS) class of pigment in many cones. During retinal development, SWS1 opsin expression generally precedes that of LWS opsin, and evidence from genetic studies indicates that the S cone pathway may be the default pathway for cone development. With the notable exception of the cartilaginous fishes, where S cones appear to be absent, they are present in representative species from all other vertebrate classes. S cone loss is not, however, uncommon; they are absent from most aquatic mammals and from some but not all nocturnal terrestrial species. The peak spectral sensitivity of S cones depends on the spectral characteristics of the pigment present. Evidence from the study of agnathans and teleost fishes indicates that the ancestral vertebrate SWS1 pigment was ultraviolet (UV) sensitive with a peak around 360 nm, but this has shifted into the violet region of the spectrum (>380 nm) on many separate occasions during vertebrate evolution. In all cases, the shift was generated by just one or a few replacements in tuning-relevant residues. Only in the avian lineage has tuning moved in the opposite direction, with the reinvention of UV-sensitive pigments.

Correlation between Nuptial Colors and Visual Sensitivities Tuned by Opsins Leads to Species Richness in Sympatric Lake Victoria Cichlid Fishes

Reproductive isolation that prevents interspecific hybridization between closely related coexisting species maintains sympatric species diversity. One of the reproductive isolations is mate choice based on color signals (breeding color perceived by color vision). This is well known in several animal taxa, yet little is known about its genetic and molecular mechanism. Lake Victoria cichlid fishes are thought to be an example of sympatric species diversity. In the species inhabiting different light environments in rocky shore, speciation by sensory drive through color signals has been proposed by analyses of the long wavelength-sensitive (LWS) opsin gene and the male nuptial coloration. However, the genetic and molecular mechanism of how diversity of sympatric species occurring in the same habitat is maintained remains unknown. To address this issue, we determined nucleotide sequences of eight opsins of six sympatric species collected from a sandy-muddy shore--an ideal model system for studying sympatric species. Among eight opsins, the LWS and RH1 alleles were diversified and one particular allele is dominant or fixed in each species, and we propose that this is due to natural selection. The functions of their LWS alleles were also diversified as shown by absorption measurements of reconstituted visual pigments. To analyze the relationship between nuptial coloration and the absorption of LWS pigments, we systematically evaluated and defined nuptial coloration. We showed that the coloration was species specific with respect to hue and significantly differentiated by the index values of hue (dominant wavelength: λ(d)). The λ(d) value of the male nuptial coloration correlated with the absorption of LWS pigments from all the species, suggesting that reproductive isolation through mate choice using color signals may prevent sympatric interspecific hybridization, thereby maintaining the species diversity in sympatric species in Lake Victoria.

Anion sensitivity and spectral tuning of middle- and long-wavelength-sensitive (MWS/LWS) visual pigments.

The long-wavelength-sensitive (LWS) opsins form one of four classes of vertebrate cone visual pigment and exhibit peak spectral sensitivities (λ(max)) that generally range from 525 to 560 nm for rhodopsin/vitamin-A(1) photopigments. Unique amongst the opsin classes, many LWS pigments show anion sensitivity through the interaction of chloride ions with a histidine residue at site 197 (H197) to give a long-wavelength spectral shift in peak sensitivity. Although it has been shown that amino acid substitutions at five sites (180, 197, 277, 285 and 308) are useful in predicting the λ(max) values of the LWS pigment class, some species, such as the elephant shark and most marine mammals, express LWS opsins that possess λ(max) values that are not consistent with this 'five-site' rule, indicating that other interactions may be involved. This study has taken advantage of the natural mutation at the chloride-binding site in the mouse LWS pigment. Through the use of a number of mutant pigments generated by site-directed mutagenesis, a new model has been formulated that takes into account the role of charge and steric properties of the side chains of residues at sites 197 and 308 in the function of the chloride-binding site in determining the peak sensitivity of LWS photopigments.

Relative LWS cone opsin expression determines optomotor thresholds in Malawi cichlid fish

Associating quantitative genetic traits with quantitative behaviors is a relatively unexplored region of sensory neurobiology. The visual system is an ideal place to test models associating these levels of sensory perception. In this study, we reared cichlid fish from Lake Malawi in different ambient light environments. We then tested the visual sensitivities of these fish using the optomotor response (OMR) behavioral paradigm and measured the relative expression of cone opsin genes. We found that the light environment experienced by fish during development can alter gene expression, particularly as it applies to the long wavelength-sensitive (LWS) opsin gene. Also, fish from different rearing conditions exhibited different behavioral sensitivities. We combined these data with predictions of opsin pigment absorption by the different OMR stimuli to determine which cone types are most likely to influence the OMR behavior. While we hypothesized that this behavior would be controlled by a random-wiring model reflecting the expression of both medium wavelength-sensitive (MWS) and LWS opsins, our models suggest that only the LWS pigment is required to predict behavior. Furthermore, analyses show that LWS expression variation accounts for ~20% of the observed behavioral variance. This work confirms that sensory gene expression influences behavior in a predictable fashion. It also suggests that the neural wiring of basal visual pathways in cichlid fish may differ from that observed in mammals and zebrafish, but is similar to that described in goldfish. This finding has important implications for the evolution of the magnocellular neural pathway in teleosts.

Intra-retinal variation of opsin gene expression in the guppy (Poecilia reticulata)

Although behavioural experiments demonstrate that colouration influences mate choice in many species, a complete understanding of this form of signalling requires information about colour vision in the species under investigation. The guppy (Poecilia reticulata) has become a model species for the study of colour-based sexual selection. To investigate the role of opsin gene duplication and divergence in the evolution of colour-based mate choice, we used in situ hybridization to determine where the guppy's nine cone opsins are expressed in the retina. Long wavelength-sensitive (LWS) opsins were more abundant in the dorsal retina than in the ventral retina. One of the middle wavelength-sensitive opsins (RH2-1) exhibited the opposite pattern, while the other middle wavelength-sensitive opsin (RH2-2) and the short wavelength-sensitive opsins (SWS1, SWS2A and SWS2B) were expressed throughout the retina. We also found variation in LWS opsin expression among individuals. These observations suggest that regions of the guppy retina are specialized with respect to wavelength discrimination and/or sensitivity. Intra-retinal variability in opsin expression, which has been observed in several fish species, might be an adaptation to variation in the strength and spectral composition of light entering the eye from above and below. The discovery that opsin expression varies in the guppy retina may motivate new behavioural experiments designed to study its role in mate choice.

In ovo thyroxine exposure alters later UVS cone loss in juvenile rainbow trout

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.

Gene Duplication and Divergence of Long Wavelength-Sensitive Opsin Genes in the Guppy, Poecilia reticulata

Female preference for male orange coloration in the genus Poecilia suggests a role for duplicated long wavelength-sensitive (LWS) opsin genes in facilitating behaviors related to mate choice in these species. Previous work has shown that LWS gene duplication in this genus has resulted in expansion of long wavelength visual capacity as determined by microspectrophotometry (MSP). However, the relationship between LWS genomic repertoires and expression of LWS retinal cone classes within a given species is unclear. Our previous study in the related species, Xiphophorus helleri, was the first characterization of the complete LWS opsin genomic repertoire in conjunction with MSP expression data in the family Poeciliidae, and revealed the presence of four LWS loci and two distinct LWS cone classes. In this study we characterized the genomic organization of LWS opsin genes by BAC clone sequencing, and described the full range of cone cell types in the retina of the colorful Cumaná guppy, Poecilia reticulata. In contrast to X. helleri, MSP data from the Cumaná guppy revealed three LWS cone classes. Comparisons of LWS genomic organization described here for Cumaná to that of X. helleri indicate that gene divergence and not duplication was responsible for the evolution of a novel LWS haplotype in the Cumaná guppy. This lineage-specific divergence is likely responsible for a third additional retinal cone class not present in X. helleri, and may have facilitated the strong sexual selection driven by female preference for orange color patterns associated with the genus Poecilia.

Spectral and polarization sensitivity of juvenile Atlantic salmon (Salmo salar): phylogenetic considerations

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.

Electrophysiological assessment of spectral sensitivity in adult Nile tilapia Oreochromis niloticus: evidence for violet sensitivity

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.

Shedding Light on Serpent Sight: The Visual Pigments of Henophidian Snakes

The biologist Gordon Walls proposed his "transmutation" theory through the 1930s and the 1940s to explain cone-like morphology of rods (and vice versa) in the duplex retinas of modern-day reptiles, with snakes regarded as the epitome of his hypothesis. Despite Walls' interest, the visual system of reptiles, and in particular snakes, has been widely neglected in favor of studies of fishes and mammals. By analyzing the visual pigments of two henophidian snakes, Xenopeltis unicolor and Python regius, we show that both species express two cone opsins, an ultraviolet-sensitive short-wavelength-sensitive 1 (SWS1) (lambda(max) = 361 nm) pigment and a long-wavelength-sensitive (LWS) (lambda(max) = 550 nm) pigment, providing the potential for dichromatic color vision. They also possess rod photoreceptors which express the usual rod opsin (Rh1) pigment with a lambda(max) at 497 nm. This is the first molecular study of the visual pigments expressed in the photoreceptors of any snake species. The presence of a duplex retina and the characterization of LWS, SWS1, and Rh1 visual pigments in henophidian snakes implies that "lower" snakes do not provide support for Walls' transmutation theory, unlike some "higher" (caenophidian) snakes and other reptiles, such as geckos. More data from other snake lineages will be required to test this hypothesis further.

The Green-absorbing Drosophila Rh6 Visual Pigment Contains a Blue-shifting Amino Acid Substitution That Is Conserved in Vertebrates

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Through sequence analysis and functional investigation of vertebrate visual pigments, numerous amino acid substitutions important for this adaptive process have been identified. Here we describe a serine/alanine (S/A) substitution in long wavelength-absorbing Drosophila visual pigments that occurs at a site corresponding to Ala-292 in bovine rhodopsin. This S/A substitution accounts for a 10-17-nm absorption shift in visual pigments of this class. Additionally, we demonstrate that substitution of a cysteine at the same site, as occurs in the blue-absorbing Rh5 pigment, accounts for a 4-nm shift. Substitutions at this site are the first spectrally significant amino acid changes to be identified for invertebrate pigments sensitive to visible light and are the first evidence of a conserved tuning mechanism in vertebrate and invertebrate pigments of this class.

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.

Retinal processing and opponent mechanisms mediating ultraviolet polarization sensitivity in rainbow trout (Oncorhynchus mykiss)

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.

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.

Emergence of Novel Color Vision in Mice Engineered to Express a Human Cone Photopigment

Changes in the genes encoding sensory receptor proteins are an essential step in the evolution of new sensory capacities. In primates, trichromatic color vision evolved after changes in X chromosome-linked photopigment genes. To model this process, we studied knock-in mice that expressed a human long-wavelength-sensitive (L) cone photopigment in the form of an X-linked polymorphism. Behavioral tests demonstrated that heterozygous females, whose retinas contained both native mouse pigments and human L pigment, showed enhanced long-wavelength sensitivity and acquired a new capacity for chromatic discrimination. An inherent plasticity in the mammalian visual system thus permits the emergence of a new dimension of sensory experience based solely on gene-driven changes in receptor organization.

Cone visual pigments of aquatic mammals

It has long been hypothesized that the visual systems of animals are evolutionarily adapted to their visual environment. The entrance many millions of years ago of mammals into the sea gave these new aquatic mammals completely novel visual surroundings with respect to light availability and predominant wavelengths. This study examines the cone opsins of marine mammals, hypothesizing, based on previous studies [Fasick et al. (1998) and Levenson & Dizon (2003)], that the deep-dwelling marine mammals would not have color vision because the pressure to maintain color vision in the dark monochromatic ocean environment has been relaxed. Short-wavelength-sensitive (SWS) and long-wavelength-sensitive (LWS) cone opsin genes from two orders (Cetacea and Sirenia) and an additional suborder (Pinnipedia) of aquatic mammals were amplified from genomic DNA (for SWS) and cDNA (for LWS) by PCR, cloned, and sequenced. All animals studied from the order Cetacea have SWS pseudogenes, whereas a representative from the order Sirenia has an intact SWS gene, for which the corresponding mRNA was found in the retina. One of the pinnipeds studied (harp seal) has an SWS pseudogene, while another species (harbor seal) appeared to have an intact SWS gene. However, no SWS cone opsin mRNA was found in the harbor seal retina, suggesting a promoter or splice site mutation preventing transcription of the gene. The LWS opsins from the different species were expressed in mammalian cells and reconstituted with the 11-cis-retinal chromophore in order to determine maximal absorption wavelengths (lambda(max)) for each. The deeper dwelling Cetacean species had blue shifted lambda(max) values compared to shallower-dwelling aquatic species. Taken together, these findings support the hypothesis that in the monochromatic oceanic habitat, the pressure to maintain color vision has been relaxed and mutations are retained in the SWS genes, resulting in pseudogenes. Additionally, LWS opsins are retained in the retina and, in deeper-dwelling animals, are blue shifted in lambda(max).

Colour vision and speciation in Lake Victoria cichlids of the genus Pundamilia

Lake Victoria cichlids are one of the most speciose groups of vertebrates. Selection on coloration is likely playing an important role in their rapid speciation. To test the hypothesis that sensory biases could explain species differences in mating preferences and nuptial coloration, we studied seven populations of four closely related species of the genus Pundamilia that differ in visual environment and male nuptial colour. Microspectrophotometry determined that the wavelength of maximum absorption (lambdamax) of the rod pigment and three cone pigments were similar in all four species. Only the long wavelength sensitive (LWS) pigment varied among species, with 3-4 nm shifts in lambdamax that correlated with differences in the LWS opsin sequence. These subtle shifts in lambdamax coincided with large shifts in male body colour, with red species having longer LWS pigments than blue species. Furthermore, we observed within and between species a correlation between water transparency and the proportion of red/red vs. red/green double cones. Individuals from turbid water had more red/red double cones than individuals from clear water. The variation in LWS lambdamax and in the proportion of red/red double cones could lead to differences in perceived brightness that may explain the evolution of variation in male coloration. However, other factors, such as chromophore shifts and higher order neural processing, should also be investigated to fully understand the physiological basis of differential responses to male mating hues in cichlid fish.

Eyeshine and spectral tuning of long wavelength-sensitive rhodopsins: no evidence for red-sensitive photoreceptors among five Nymphalini butterfly species

Spectral tuning of rhodopsins commonly refers to the effects of opsin amino acid substitutions on the wavelength for peak sensitivity of the rhodopsin absorption spectrum. Nymphalini butterflies provide an opportunity for identifying some of the amino acid substitutions responsible for insect rhodopsin spectral tuning because the majority of photoreceptor cells (R3-9) in the adult retina express only a single long wavelength-sensitive (LWS) opsin mRNA transcript. Therefore, the opsin genotype can be directly correlated with its phenotype. We determined the LWS opsin gene sequence from cDNA of the mourning cloak Nymphalis antiopa, and from genomic DNA of the malachite Siproeta stelenes and the peacock Inachis io. Using an epi-microspectrophotometer we examined each butterfly's eyeshine for photochemical evidence of multiple LWS rhodopsins and found only one. We then performed partial-bleaching experiments to obtain absorbance spectra for the LWS rhodopsins of all three species as well as from another nymphalid, the buckeye Junonia coenia. The isolated LWS opsin gene sequences varied in length from 1437-1612 bp and encode rhodopsins R522 (S. stelenes), R530 (I. io), R534 (N. antiopa) and, together with a previously published sequence, R510 (J. coenia). Comparative sequence analysis indicates that the S. stelenes rhodopsin is slightly blue-shifted compared to the typical 530 nm lepidopteran rhodopsin because of the presence of a S138A substitution at a homologous site that in mammalian MWS/LWS rhodopsins causes a 5 nm blue-shift. The difference in peak absorption between R522 of S. stelenes and R530 of Inachis io is therefore largely accounted for by this substitution. This suggests that spectral tuning mechanisms employing the S138A may have evolved in parallel in mammalian and butterfly MWS/LWS rhodopsins across 500 million years of evolution.

Visual sensitivity across the menstrual cycle

This study was designed to evaluate the hypothesis that hormonal change can affect lower level light-adaptation processes, which are likely to be retinally based. Foveal visual sensitivities were measured across several menstrual cycles of four women not using hormonally acting medication and across several menstrual cycles of three women using a triphasic oral contraceptive. One woman, diagnosed with premenstrual syndrome (PMS), was a subject for both groups. Sensitivities were measured for a series of test wavelengths for 580-nm backgrounds of 2.0 and 4.0 log td. Of the six individuals tested, one had clear evidence of visual-adaptation changes occurring in phase with the menstrual cycle. Prior to using the oral contraceptive, this individual (the PMS subject) experienced changes of short-wavelength-sensitive (SWS)-cone-mediated sensitivities of up to about 1.4 log unit on the 4.0 log td background. Her SWS-cone-mediated sensitivities tended to be highest near ovulation and lowest premenstrually. Threshold-versus-illuminance (TVI) curves confirmed that the rate of sensitivity decrease with increasing background illuminance (i.e. the TVI slope) was greater premenstrually. The degree of background-induced desensitization within her middle-wavelength-sensitive (MWS)/long-wavelength-sensitive (LWS) cone pathways also appeared to vary cyclically, but the magnitude of the variation was smaller and the time course appeared to be different. When this subject began oral contraceptive use, the patterns of sensitivity change were all altered. None of the other five subjects experienced changes of SWS-cone-mediated vision that were cyclic and significantly adaptation-state dependent. However, there was evidence for a limited degree of cyclic adaptation change within the MWS/LWS cone pathways of at least one additional subject. We conclude that hormonal change can--for some unknown proportion of women--be linked to alterations of retinal function. However, the alterations are not the same for all visual pathways, and there are pronounced individual differences. The data also demonstrate that individuals' visual adaptation capabilities can vary substantially over periods of weeks.

Early Duplication and Functional Diversification of the Opsin Gene Family in Insects

Recent analysis of the complete mosquito Anopheles gambiae genome has revealed a far higher number of opsin genes than for either the Drosophila melanogaster genome or any other known insect. In particular, the analysis revealed an extraordinary opsin gene content expansion, whereby half are long wavelength-sensitive (LW) opsin gene duplicates. We analyzed this genomic data in relationship to other known insect opsins to estimate the relative timing of the LW opsin gene duplications and to identify "missing" paralogs in extant species. The inferred branching patterns of the LW opsin gene family phylogeny indicate at least one early gene duplication within insects before the emergence of the orders Orthoptera, Mantodea, Hymenoptera, Lepidoptera, and Diptera. These data predict the existence of one more LW opsin gene than is currently known from most insects. We tested this prediction by using a degenerate PCR strategy to screen the hymenopteran genome for novel LW opsin genes. We isolated two LW opsin gene sequences from each of five bee species, Bombus impatiens, B. terrestris, Diadasia afflicta, D. rinconis, and Osmia rufa, including 1.1 to 1.2 kb from a known (LW Rh1) and 1 kb from a new opsin gene (LW Rh2). Phylogenetic analysis suggests that the novel hymenopteran gene is orthologous to A. gambiae GPRop7, a gene that is apparently missing from D. melanogaster. Relative rate tests show that LW Rh2 is evolving at a slower rate than LW Rh1 and, therefore, may be a useful marker for higher-level hymenopteran systematics. Site-specific rate tests indicate the presence of several amino acid sites between LW Rh1 and LW Rh2 that have undergone shifts in selective constraints after duplication. These sites and others are discussed in relationship to putative structural and functional differences between the two genes.