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.     

Cone opsin genes of african cichlid fishes: tuning spectral sensitivity by differential gene expression

Spectral tuning of visual pigments is typically accomplished through changes in opsin amino acid sequence. Within a given opsin class, changes at a few key sites control wavelength specificity. To investigate known differences in the visual pigment spectral sensitivity of the Lake Malawi cichlids, Metriaclima zebra (368, 488, and 533 nm) and Dimidiochromis compressiceps (447, 536, and 569 nm), we sequenced cone opsin genes from these species as well as Labeotropheus fuelleborni and Oreochromis niloticus. These cichlids have five distinct classes of cone opsin genes, including two unique SWS-2 genes. Comparisons of the inferred amino acid sequences from the five cone opsin genes of M. zebra, D. compressiceps, and L. fuelleborni show the sequences to be nearly identical. Therefore, evolution of key opsin sites cannot explain the differences in visual pigment sensitivities. Real-time PCR demonstrates that different cichlid species express different subsets of the available cone opsin genes. Metriaclima zebra and L. fuelleborni express a complement of genes which give them UV-shifted visual pigments, while D. compressiceps expresses a different set to produce a red-shifted visual system. Thus, variations in cichlid spectral sensitivity have arisen through evolution of gene regulation, rather than through changes in opsin amino acid sequence.     

Intraspecific cone opsin expression variation in the cichlids of Lake Malawi

The expression of cone opsin genes is a primary determinant of the characteristics of colour vision. Interspecific variation in opsin expression is common in African cichlids. It is correlated with foraging among cichlids from Lake Malawi, and with ambient light environment among cichlids from Lake Victoria. In this study, we tested whether gene expression varied within species such that it might be important in contributing to divergence. We hypothesized that light attenuation with depth would be correlated with predictable changes in gene expression in Lake Malawi, and that this variation would tune visual sensitivities to match the ambient light environment. We observed significant differences in cone opsin expression in three different comparisons among populations of the same species. Higher LWS expression was found in shallow versus deep Copadichromis eucinostomus. In Metriaclima zebra, individuals from Zimbawe Rock expressed significantly more SWS2B than those from Thumbi West Island, although these locales have similar ambient light environments. Finally, Tropheops gracilior from deeper water had significantly more variation in expression than their shallow counterparts. These results support that gene expression varies significantly between populations of the same species. Surprisingly, these results could not be explained by predicted visual performance as models predicted that differential expression patterns did not confer sensitivity advantages at different depths. This suggested that expression variation did not confer a local sensitivity advantage. Therefore, our findings were contrary to a primary requirement of the sensory bias hypothesis. As such, other explanations for intraspecific gene expression variation need to be tested.     

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.     

Proximate and ultimate causes of variable visual sensitivities: Insights from cichlid fish radiations

Animals vary in their sensitivities to different wavelengths of light. Sensitivity differences can have fitness implications in terms of animals' ability to forage, find mates, and avoid predators. As a result, visual systems are likely selected to operate in particular lighting environments and for specific visual tasks. This review focuses on cichlid vision, as cichlids have diverse visual sensitivities, and considerable progress has been made in determining the genetic basis for this variation. We describe both the proximate and ultimate mechanisms shaping cichlid visual diversity using the structure of Tinbergen's four questions. We describe (1) the molecular mechanisms that tune visual sensitivities including changes in opsin sequence and expression; (2) the evolutionary history of visual sensitivity across the African cichlid flocks; (3) the ontological changes in visual sensitivity and how modifying this developmental program alters sensitivities among species; and (4) the fitness benefits of spectral tuning mechanisms with respect to survival and mating success. We further discuss progress to unravel the gene regulatory networks controlling opsin expression and suggest that a simple genetic architecture contributes to the lability of opsin gene expression. Finally, we identify unanswered questions including whether visual sensitivities are experiencing selection, and whether similar spectral tuning mechanisms shape visual sensitivities of other fishes. genesis 54:299–325, 2016. © 2016 Wiley Periodicals, Inc.     

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.     

Genetic basis of differential opsin gene expression in cichlid fishes

Visual sensitivity can be tuned by differential expression of opsin genes. Among African cichlid fishes, seven cone opsin genes are expressed in different combinations to produce diverse visual sensitivities. To determine the genetic architecture controlling these adaptive differences, we analysed genetic crosses between species expressing different complements of opsin genes. Quantitative genetic analyses suggest that expression is controlled by only a few loci with correlations among some genes. Genetic mapping identifies clear evidence of trans‐acting factors in two chromosomal regions that contribute to differences in opsin expression as well as one cis‐regulatory region. Therefore, both cis and trans regulation are important. The simple genetic architecture suggested by these results may explain why opsin gene expression is evolutionarily labile, and why similar patterns of expression have evolved repeatedly in different lineages.     

Allelic Variation in Malawi Cichlid Opsins: A Tale of Two Genera

The role of sequence variation in the spectral tuning of color vision is well established in many systems. This includes the cichlids of Lake Victoria where sequence variation has been linked to environmental light gradients and speciation. The cichlids of Lake Malawi are a similar model for visual evolution, but the role of gene sequence variation in visual tuning between closely related species is unknown. This work describes such variation in multiple species of two rock-dwelling genera: Metriaclima and Labidochromis. Genomic DNA for seven cone opsin genes was sequenced and the structure of the opsin proteins was inferred. Retinal binding pocket polymorphisms were identified and compared to available data regarding spectral absorbance shifts. Sequence variation with known or potential effects on absorbance spectra were found in four genes: SWS1 (UV sensitive), SWS2B (violet sensitive), RH2Aβ (green sensitive), and LWS (red sensitive). Functional variation was distributed such that each genus had both a variable short-wavelength and long-wavelength sensitive opsin. This suggests spectral tuning is important at the margins of the cichlid visual spectrum. Further, there are two SWS1 opsin alleles that differ in sensitivity by 10 nm and are >2 MY divergent. One of these occurs in a haplotype block >1 kb. Potential haplotype blocks were found around the RH2 opsin loci. These data suggest that molecular diversification has resulted in functionally unique alleles and changes to the visual system. These data also suggest that opsin sequence variation tunes spectral sensitivities between closely related species and that the specific regions of spectral tuning are genus-specific.     

Evolution of opsin expression in birds driven by sexual selection and habitat

Theories of sexual and natural selection predict coevolution of visual perception with conspecific colour and/or the light environment animals occupy. One way to test these theories is to focus on the visual system, which can be achieved by studying the opsin-based visual pigments that mediate vision. Birds vary greatly in colour, but opsin gene coding sequences and associated visual pigment spectral sensitivities are known to be rather invariant across birds. Here, I studied expression of the four cone opsin genes (Lws, Rh2, Sws2 and Sws1) in 16 species of New World warblers (Parulidae). I found levels of opsin expression vary both across species and between the sexes. Across species, female, but not male Sws2 expression is associated with an index of sexual selection, plumage dichromatism. This fits predictions of classic sexual selection models, in which the sensory system changes in females, presumably impacting female preference, and co-evolves with male plumage. Expression of the opsins at the extremes of the light spectrum, Lws and Uvs, correlates with the inferred light environment occupied by the different species. Unlike opsin spectral tuning, regulation of opsin gene expression allows for fast adaptive evolution of the visual system in response to natural and sexual selection, and in particular, sex-specific selection pressures.     

Gene duplication and differential gene expression play an important role in the diversification of visual pigments in fish

Sensory systems provide crucial information about an organism's external environment and, thus, are often subject to strong natural selection. Because of the large variation in the intensity and spectral quality of light in aquatic environments, studies of sensory adaptation have focused on the visual systems of fish for over a half a century. Recently, the molecular genetic mechanisms that determine the spectral sensitivity of visual pigments have been characterized in several fishes including zebrafish, guppies, medaka, killifish, bream, and cichlids. The results of these studies suggest that teleost fish have incredibly diverse visual systems. In this paper, we review the role that opsin duplication and differential gene expression have played in the diversification of visual pigments. We compare our findings in cichlids to five other taxonomic groups and highlight the ways that their similarities and differences may provide new insights into the molecular genetic basis of sensory adaptation and diversification.     

Mix and match color vision: tuning spectral sensitivity by differential opsin gene expression in Lake Malawi cichlids

Cichlid fish of the East African Rift Lakes are renowned for their diversity and offer a unique opportunity to study adaptive changes in the visual system in rapidly evolving species flocks. Since color plays a significant role in mate choice, differences in visual sensitivities could greatly influence and even drive speciation of cichlids. Lake Malawi cichlids inhabiting rock and sand habitats have significantly different cone spectral sensitivities. By combining microspectrophotometry (MSP) of isolated cones, sequencing of opsin genes, and spectral analysis of recombinant pigments, we have established the cone complements of four species of Malawi cichlids. MSP demonstrated that each of these species predominately expresses three cone pigments, although these differ between species to give three spectrally different cone complements. In addition, rare populations of spectrally distinct cones were found. In total, seven spectral classes were identified. This was confirmed by opsin gene sequencing, expression, and in vitro reconstitution. The genes represent the four major classes of cone opsin genes that diverged early in vertebrate evolution. All four species possess a long-wave-sensitive (LWS), three spectrally distinct green-sensitive (RH2), a blue-sensitive (SWS2A), a violet-sensitive (SWS2B), and an ultraviolet-sensitive (SWS1) opsin. However, African cichlids determine their spectral sensitivity by differential expression of primarily only three of the seven available cone opsin genes. Phylogenetic analysis suggests that all percomorph fish have similar potential.     

Adult plasticity in African cichlids: Rapid changes in opsin expression in response to environmental light differences

Phenotypic plasticity allows organisms to adapt quickly to local environmental conditions and could facilitate adaptive radiations. Cichlids have recently undergone an adaptive radiation in Lake Malawi where they inhabit diverse light environments and tune their visual sensitivity through differences in cone opsin expression. While cichlid opsin expression is known to be plastic over development, whether adults remain plastic is unknown. Adult plasticity in visual tuning could play a role in cichlid radiations by enabling survival in changing environments and facilitating invasion into novel environments. Here we examine the existence of and temporal changes in adult visual plasticity of two closely related species. In complementary experiments, wild adult Metriaclima mbenji from Lake Malawi were moved to the lab under UV‐deficient fluorescent lighting; while lab raised M. benetos were placed under UV‐rich lighting designed to mimic light conditions in the wild. Surprisingly, adult cichlids in both experiments showed significant changes in the expression of the UV‐sensitive single cone opsin, SWS1, in only 3 days. Modeling quantum catches in the light environments revealed a possible link between the light available to the SWS1 visual pigment and SWS1 expression. We conclude that adult cichlids can undergo rapid and significant changes in opsin expression in response to environmental light shifts that are relevant to their habitat and evolutionary history in Lake Malawi. This could have contributed to the rapid divergence characteristic of these fantastic fishes.     

Rapid adaptation to a novel light environment: The importance of ontogeny and phenotypic plasticity in shaping the visual system of Nicaraguan Midas cichlid fish (Amphilophus citrinellus spp.)

Colonization of novel habitats is typically challenging to organisms. In the initial stage after colonization, approximation to fitness optima in the new environment can occur by selection acting on standing genetic variation, modification of developmental patterns or phenotypic plasticity. Midas cichlids have recently colonized crater Lake Apoyo from great Lake Nicaragua. The photic environment of crater Lake Apoyo is shifted towards shorter wavelengths compared to great Lake Nicaragua and Midas cichlids from both lakes differ in visual sensitivity. We investigated the contribution of ontogeny and phenotypic plasticity in shaping the visual system of Midas cichlids after colonizing this novel photic environment. To this end, we measured cone opsin expression both during development and after experimental exposure to different light treatments. Midas cichlids from both lakes undergo ontogenetic changes in cone opsin expression, but visual sensitivity is consistently shifted towards shorter wavelengths in crater lake fish, which leads to a paedomorphic retention of their visual phenotype. This shift might be mediated by lower levels of thyroid hormone in crater lake Midas cichlids (measured indirectly as dio2 and dio3 gene expression). Exposing fish to different light treatments revealed that cone opsin expression is phenotypically plastic in both species during early development, with short and long wavelength light slowing or accelerating ontogenetic changes, respectively. Notably, this plastic response was maintained into adulthood only in the derived crater lake Midas cichlids. We conclude that the rapid evolution of Midas cichlids’ visual system after colonizing crater Lake Apoyo was mediated by a shift in visual sensitivity during ontogeny and was further aided by phenotypic plasticity during development.     

Molecular cloning of cDNA encoding red opsin gene in the retinas of five Antarctic notothenioid fishes

Previous evidence suggested that notothenioid fish had lost red-sensitive (LWS) visual pigment and photoreceptors, but retained ultraviolet-sensitive (SWS1), blue-sensitive (SWS2), and green-sensitive (RH2) pigments. We used RT-PCR and Southern blot to isolate the LWS opsin gene in five notothenioid species. We determined full-coding LWS opsin sequences and genomic sequences. The expected peak absorbance of the LWS opsin, based on the five-sites rule that is primarily responsible for the spectral sensitivities in vertebrates, ranged from 541 to 553 nm. In Antarctic waters, light of this wavelength penetrates to dozens of meters. Thus, we conclude that notothenioids use tetrachromatic color vision in shallower waters, at least during the Antarctic summer.     

Conserved visual sensitivities across divergent lizard lineages that differ in an ultraviolet sexual signal

The sensory drive hypothesis predicts the correlated evolution of signaling traits and sensory perception in differing environments. For visual signals, adaptive divergence in both color signals and visual sensitivities between populations may contribute to reproductive isolation and promote speciation, but this has rarely been tested or shown in terrestrial species. We tested whether opsin protein expression differs between divergent lineages of the tawny dragon (Ctenophorus decresii) that differ in the presence/absence of an ultraviolet sexual signal. We measured the expression of four retinal cone opsin genes (SWS1, SWS2, RH2, and LWS) using droplet digital PCR. We show that gene expression between lineages does not differ significantly, including the UV wavelength sensitive SWS1. We discuss these results in the context of mounting evidence that visual sensitivities are highly conserved in terrestrial systems. Multiple competing requirements may constrain divergence of visual sensitivities in response to sexual signals. Instead, signal contrast could be increased via alternative mechanisms, such as background selection. Our results contribute to a growing understanding of the roles of visual ecology, phylogeny, and behavior on visual system evolution in reptiles.     

Estimated absorbance spectra of the visual pigments of the North Atlantic right whale (Eubalaena glacialis)

To assess the spectral sensitivities of the retinal visual pigments from the North Atlantic right whale (Eubalaena glacialis), we have cloned and sequenced two exons from the rod opsin gene and two exons from the middle‐wavelength sensitive (MWS) cone opsin gene in order to determine the amino acids at positions known to be key regulators of the spectral location of the absorbance maximum (λ_max). Based on previous mutagenesis models we estimate that the right whale possesses a rod visual pigment with a λ_max of 499 nm and a MWS cone visual pigment with a λ_max of 524 nm. Although the MWS cone visual pigment from the right whale is blue‐shifted in its spectral sensitivity like those from odontocetes, the spectral sensitivity of the right whale rod visual pigment is similar to those from terrestrial mammals.     

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.     

The Drosophila ninaE gene encodes an opsin

The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.