Rapid light-induced shifts in opsin expression: finding new opsins, discerning mechanisms of change, and implications for visual sensitivity

Light-induced shifts in cone frequency and opsin expression occur in many aquatic species. Yet little is known about how quickly animals can alter opsin expression and, thereby, track their visual environments. Similarly, little is known about whether adult animals can alter opsin expression or whether shifts in opsin expression are limited to critical developmental windows. We took adult wild-caught bluefin killifish (Lucania goodei) from three different lighting environments (spring, swamp and variable), placed them under two different lighting treatments (clear vs. tea-stained water) and monitored opsin expression over 4 weeks. We measured opsin expression for five previously described opsins (SWS1, SWS2B, SWS2A, RH2-1 and LWS) as well as RH2-2 which we discovered via 454 sequencing. We used two different metrics of opsin expression. We measured expression of each opsin relative to a housekeeping gene and the proportional expression of each opsin relative to the total pool of opsins. Population and lighting environment had large effects on opsin expression which were present at the earliest time points indicating rapid shifts in expression. The two measures of expression produced radically different patterns. Proportional measures indicated large effects of light on SWS1 expression, whereas relative measures indicated no such effect. Instead, light had large effects on the relative expression of SWS2B, RH2-2, RH2-1 and LWS. We suggest that proportional measures of opsin expression are best for making inferences about colour vision, but that measures relative to a housekeeping gene are better for making conclusions about which opsins are differentially regulated.     

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.     

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

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

Rods are rods and cones cones, and (never) the twain shall meet.

More than 100 photopigment G protein-coupled receptors (opsins) have been sequenced and organized into six classes. Rod photoreceptors in various species have been found to express an opsin from one of the two rhodopsin classes, while cones express an opsin from one of the four remaining classes. It has now been discovered that salamander short-wavelength sensitive cones and green rods express the same opsin, while manifesting other features that classically distinguish rods from cones.     

Signatures of selection and gene conversion associated with human color vision variation

Trichromatic color vision in humans results from the combination of red, green, and blue photopigment opsins. Although color vision genes have been the targets of active molecular and psychophysical research on color vision abnormalities, little is known about patterns of normal genetic variation in these genes among global human populations. The current study presents nucleotide sequence analyses and tests of neutrality for a 5.5-kb region of the X-linked long-wave "red" opsin gene (OPN1LW) in 236 individuals from ethnically diverse human populations. Our analysis of the recombination landscape across OPN1LW reveals an unusual haplotype structure associated with amino acid replacement variation in exon 3 that is consistent with gene conversion. Compared with the absence of OPN1LW amino acid replacement fixation since divergence from chimpanzee, the human population exhibits a significant excess of high-frequency OPN1LW replacements. Our results suggest that subtle changes in L-cone opsin wavelength absorption may have been adaptive during human evolution.     

Depth‐dependent plasticity in opsin gene expression varies between damselfish (Pomacentridae) species

Phenotypic plasticity plays an important role in adapting the visual capability of many animal species to changing sensory requirements. Such variability may be driven by developmental change or may result from environmental changes in light habitat, thereby improving performance in different photic environments. In this study, we examined inter‐ and intraspecific plasticity of visual sensitivities in seven damselfish species, part of the species‐rich and colourful fish fauna of the Great Barrier Reef in Australia. Our goal was to test whether the visual systems of damselfish were tuned to the prevailing light environment in different habitats and/or other aspects of their lifestyle. More specifically, we compared the opsin gene expression levels from individuals living in different photic habitats. We found that all species expressed rod opsin (RH1) used for dim‐light vision, and primarily three cone opsins (SWS1, RH2B and RH2A) used for colour vision. While RH1 levels changed exclusively following a diurnal cycle, cone opsin expression varied with depth in four of the seven species. Estimates of visual pigment performance imply that changes in opsin expression adjust visual sensitivities to the dominant photic regime. However, we also discovered that some species show a more stable opsin expression profile. Further, we found indication that seasonal changes, possibly linked to changes in the photic environment, might also trigger opsin expression. These findings suggest that plasticity in opsin gene expression of damselfish is highly species‐specific, possibly due to ecological differences in visual tasks or, alternatively, under phylogenetic constraints.     

Testing sensory drive speciation in cichlid fish: Linking light conditions to opsin expression,opsin genotype and female mate preference

Ecological speciation is facilitated when divergent adaptation has direct effects on selective mating. Divergent sensory adaptation could generate such direct effects, by mediating both ecological performance and mate selection. In aquatic environments, light attenuation creates distinct photic environments, generating divergent selection on visual systems. Consequently, divergent sensory drive has been implicated in the diversification of several fish species. Here, we experimentally test whether divergent visual adaptation explains the divergence of mate preferences in Haplochromine cichlids. Blue and red Pundamilia co‐occur across south‐eastern Lake Victoria. They inhabit different photic conditions and have distinct visual system properties. Previously, we documented that rearing fish under different light conditions influences female preference for blue versus red males. Here, we examine to what extent variation in female mate preference can be explained by variation in visual system properties, testing the causal link between visual perception and preference. We find that our experimental light manipulations influence opsin expression, suggesting a potential role for phenotypic plasticity in optimizing visual performance. However, variation in opsin expression does not explain species differences in female preference. Instead, female preference covaries with allelic variation in the long‐wavelength‐sensitive opsin gene (LWS), when assessed under broad‐spectrum light. Taken together, our study presents evidence for environmental plasticity in opsin expression and confirms the important role of colour perception in shaping female mate preferences in Pundamilia. However, it does not constitute unequivocal evidence for the direct effects of visual adaptation on assortative mating.     

Differential Expression of Duplicated Opsin Genes in Two EyeTypes of Ostracod Crustaceans

In the first molecular study of ostracod (Crustacea) vision, we present partial cDNA sequences of ostracod visual pigment genes (opsins). We found strong support for differential expression of opsins in ostracod median and compound eyes and suggest that photoreceptor specific expression may be a general phenomenon in organisms with multiple receptors. We infer that eye-specific expression predates the divergence of the two species examined, Skogsbergia lerneri and Vargula hilgendorfii, because eye-specific opsin orthologs are present in both species. We found multiple opsin loci in ostracods, estimating that at least eight are present in Skogsbergia lerneri. All opsins from both ostracod species examined are more closely related to each other than to any other known opsin sequences. Because we find no evidence for gene conversion or alternative splicing, we suggest the occurrence of many recent gene duplications. Why ostracods may have retained multiple recent opsin gene duplicates is unknown, but we discuss several possible hypotheses.[Reviewing Editor: Martin Kreitman]     

Population variation in opsin expression in the bluefin killifish,<Emphasis Type="Italic"> Lucania goodei</Emphasis>: a real-time PCR study

Quantitative genetics have not been used in vision studies because of the difficulty of objectively measuring large numbers of individuals. Here, we examine the effectiveness of a molecular technique, real-time PCR, as an inference of visual components in the bluefin killifish, Lucania goodei, to determine whether there is population variation in opsin expression. Previous work has shown that spring animals possess a higher frequency of UV and violet cones and a lower frequency of yellow and red cones than swamp animals. Here, we found a good qualitative match between the population differences in opsin expression and those found previously in cone frequency. Spring animals expressed higher amounts of SWS1 and SWS2B opsins (which correspond to UV and violet photopigments) and lower amounts of RH2 and LWS opsins (which correspond to yellow and red photopigments) than swamp animals. The counterintuitive pattern between color pattern, lighting environment, and vision remains. Males with blue anal fins are more abundant in swamps where animals express fewer SWS1 and SWS2B opsins and where transmission of UV/blue wavelengths is low. Understanding this system requires quantitative genetic studies. Real-time PCR is an effective tool for studies requiring inferences of visual physiology in large numbers of individuals.Abbreviations ERG electroretinogram - MSP microspectrophotometry     

Plasticity of opsin gene expression in cichlids from Lake Malawi

Sensory systems play crucial roles in survival and reproduction. Therefore, sensory plasticity has important evolutionary implications. In this study, we examined retinal plasticity in five species of cichlid fish from Lake Malawi. We compared the cone opsin expression profiles of wild‐caught fish to lab‐reared F₁ that had been raised in a UV minus, reduced intensity light environment. All of the opsin genes that were expressed in wild‐caught fish were also expressed in lab‐reared individuals. However, we found statistically significant differences in relative opsin expression among all five species. The most consistent difference was in the SWS2B (violet) opsin, which was always expressed at higher levels in lab‐reared individuals. Estimates of visual pigment quantum catch suggest that this change in expression would increase retinal sensitivity in the light environment of the lab. We also found that the magnitude of plasticity varied across species. These findings have important implications for understanding the genetic regulation of opsin expression and raise many interesting questions about how the cichlid visual system develops. They also suggest that sensory plasticity may have facilitated the ecological diversification of cichlids in Lake Malawi.     

Molecular characterization of crustacean visual pigments and the evolution of pancrustacean opsins

Investigations of opsin evolution outside of vertebrate systems have long been focused on insect visual pigments, whereas other groups have received little attention. Furthermore, few studies have explicitly investigated the selective influences across all the currently characterized arthropod opsins. In this study, we contribute to the knowledge of crustacean opsins by sequencing 1 opsin gene each from 6 previously uncharacterized crustacean species (Euphausia superba, Homarus gammarus, Archaeomysis grebnitzkii, Holmesimysis costata, Mysis diluviana, and Neomysis americana). Visual pigment spectral absorbances were measured using microspectrophotometry for species not previously characterized (A. grebnitzkii=496 nm, H. costata=512 nm, M. diluviana=501 nm, and N. americana=520 nm). These novel crustacean opsin sequences were included in a phylogenetic analysis with previously characterized arthropod opsin sequences to determine the evolutionary placement relative to the well-established insect spectral clades (long-/middle-/short-wavelength sensitive). Phylogenetic analyses indicate these novel crustacean opsins form a monophyletic clade with previously characterized crayfish opsin sequences and form a sister group to insect middle-/long-wavelength-sensitive opsins. The reconstructed opsin phylogeny and the corresponding spectral data for each sequence were used to investigate selective influences within arthropod, and mainly "pancrustacean," opsin evolution using standard dN/dS ratio methods and more sensitive techniques investigating the amino acid property changes resulting from nonsynonymous replacements in a historical (i.e., phylogenetic) context. Although the conservative dN/dS methods did not detect any selection, 4 amino acid properties (coil tendencies, compressibility, power to be at the middle of an alpha-helix, and refractive index) were found to be influenced by destabilizing positive selection. Ten amino acid sites relating to these properties were found to face the binding pocket, within 4 A of the chromophore and thus have the potential to affect spectral tuning.     

New evidence for the role of heterochrony in the repeated evolution of cichlid opsin expression

SUMMARY Lake Malawi (LM) cichlids have undergone heterochronic shifts in the expression of their cone opsin genes, the genes responsible for color vision. These shifts have generated species with short-, middle-, and long-wavelength-sensitive cone photoreceptors and visual systems. However, it is unclear when during the evolution of African cichlids these shifts occurred, or whether they could account for similar short- and middle-wavelength-sensitive profiles among unrelated cichlids in Lake Tanganyika (LT). To address these questions, we surveyed opsin expression in developing fry of two African cichlids, Astatotilapia burtoni from LT and Melanochromis auratus from LM. We found that A. burtoni expresses a series of three different single-cone opsins over the course of development, while M. auratus exhibits variation in the expression of only two. Neither A. burtoni nor M. auratus exhibits much variation in the expression of its double-cone opsins. These patterns reveal that A. burtoni exhibits progressive development in the sensitivity of its single-cone photoreceptors, but direct development in the sensitivity of its double-cone photoreceptors. M. auratus exhibits neotenic development in the sensitivity of both photoreceptor sets. Given the intermediate phylogenetic placement of A. burtoni between cichlids from LT and LM, our results suggest that the ancestor of LM's cichlids exhibited a progressive developmental pattern of opsin expression. These results indicate that the heterochronic shifts which produced the short- and middle-wavelength-sensitive profiles of LM's cichlids occurred recently, and suggest that the presence of similar profiles among LT's cichlids are due to parallel heterochronic shifts.     

Functional characterization of visual opsin repertoire in Medaka (Oryzias latipes)

A variety of visual pigment repertoires present in fish species is believed due to the great variation under the water of light environment. A complete set of visual opsin genes has been isolated and characterized for absorption spectra and expression in the retina only in zebrafish. Medaka (Oryzias latipes) is a fish species phylogenetically distant from zebrafish and has served as an important vertebrate model system in molecular and developmental genetics. We previously isolated a medaka rod opsin gene (RH1). In the present study we isolated all the cone opsin genes of medaka by genome screening of a lambda-phage and bacterial artificial chromosome (BAC) libraries. The medaka genome contains two red, LWS-A and LWS-B, three green, RH2-A, RH2-B and RH2-C, and two blue, SWS2-A and SWS2-B, subtype opsin genes as well as a single-copy of the ultraviolet, SWS1, opsin gene. Previously only one gene was believed present for each opsin type as reported in a cDNA-based study. These subtype opsin genes are closely linked and must be the products of local gene duplications but not of a genome-wide duplication. Peak absorption spectra (lambda(max)) of the reconstituted photopigments with 11-cis retinal varied greatly among the three green opsins, 452 nm for RH2-A, 516 nm for RH2-B and 492 nm for RH2-C, and between the two blue opsins, 439 nm for SWS2-A and 405 nm for SWS2-B. Zebrafish also has multiple opsin subtypes, but phylogenetic analysis revealed that medaka and zebrafish gained the subtype opsins independently. The lambda and BAC DNA clones isolated in this study could be useful for investigating the regulatory mechanisms and evolutionary diversity of fish opsin genes.     

The Evolution and Expression of the Moth Visual Opsin Family

Because visual genes likely evolved in response to their ambient photic environment, the dichotomy between closely related nocturnal moths and diurnal butterflies forms an ideal basis for investigating their evolution. To investigate whether the visual genes of moths are associated with nocturnal dim-light environments or not, we cloned long-wavelength (R), blue (B) and ultraviolet (UV) opsin genes from 12 species of wild-captured moths and examined their evolutionary functions. Strong purifying selection appeared to constrain the functions of the genes. Dark-treatment altered the levels of mRNA expression in Helicoverpa armigera such that R and UV opsins were up-regulated after dark-treatment, the latter faster than the former. In contrast, B opsins were not significantly up-regulated. Diel changes of opsin mRNA levels in both wild-captured and lab-reared individuals showed no significant fluctuation within the same group. However, the former group had significantly elevated levels of expression compared with the latter. Consequently, environmental conditions appeared to affect the patterns of expression. These findings and the proportional expression of opsins suggested that moths potentially possessed color vision and the visual system played a more important role in the ecology of moths than previously appreciated. This aspect did not differ much from that of diurnal butterflies.