Evolution of rhodopsin in flatfishes (Pleuronectiformes) is associated with depth and migratory behavior.

Visual signals are involved in many fitness-related tasks and are therefore essential for survival in many species. Aquatic organisms are ideal systems to study visual evolution, as the high diversity of spectral properties in aquatic environments generates great potential for adaptation to different light conditions. Flatfishes are an economically important group, with over 800 described species distributed globally, including halibut, flounder, sole, and turbot. The diversity of flatfish species and wide array of environments they occupy provides an excellent opportunity to understand how this variation translates to molecular adaptation of vision genes. Using models of molecular evolution, we investigated how the light environments inhabited by different flatfish lineages have shaped evolution in the rhodopsin gene, which is responsible for mediating dim-light visual transduction. We found strong evidence for positive selection in rhodopsin, and this was correlated with both migratory behavior and several fundamental aspects of habitat, including depth and freshwater/marine evolutionary transitions. We also identified several mutations that likely affect the wavelength of peak absorbance of rhodopsin, and outline how these shifts in absorbance correlate with the response to the light spectrum present in different habitats. This is the first study of rhodopsin evolution in flatfishes that considers their extensive diversity, and our results highlight how ecologically-driven molecular adaptation has occurred across this group in response to transitions to novel light environments.

Sampling Strategies for Experimentally Mapping Molecular Fitness Landscapes Using High-Throughput Methods.

Empirical studies of genotype-phenotype-fitness maps of proteins are fundamental to understanding the evolutionary process, in elucidating the space of possible genotypes accessible through mutations in a landscape of phenotypes and fitness effects. Yet, comprehensively mapping molecular fitness landscapes remains challenging since all possible combinations of amino acid substitutions for even a few protein sites are encoded by an enormous genotype space. High-throughput mapping of genotype space can be achieved using large-scale screening experiments known as multiplexed assays of variant effect (MAVEs). However, to accommodate such multi-mutational studies, the size of MAVEs has grown to the point where a priori determination of sampling requirements is needed. To address this problem, we propose calculations and simulation methods to approximate minimum sampling requirements for multi-mutational MAVEs, which we combine with a new library construction protocol to experimentally validate our approximation approaches. Analysis of our simulated data reveals how sampling trajectories differ between simulations of nucleotide versus amino acid variants and among mutagenesis schemes. For this, we show quantitatively that marginal gains in sampling efficiency demand increasingly greater sampling effort when sampling for nucleotide sequences over their encoded amino acid equivalents. We present a new library construction protocol that efficiently maximizes sequence variation, and demonstrate using ultradeep sequencing that the library encodes virtually all possible combinations of mutations within the experimental design. Insights learned from our analyses together with the methodological advances reported herein are immediately applicable toward pooled experimental screens of arbitrary design, enabling further assay upscaling and expanded testing of genotype space.

Multiple Ecological Axes Drive Molecular Evolution of Cone Opsins in Beloniform Fishes.

Ecological and evolutionary transitions offer an excellent opportunity to examine the molecular basis of adaptation. Fishes of the order Beloniformes include needlefishes, flyingfishes, halfbeaks, and allies, and comprise over 200 species occupying a wide array of habitats-from the marine epipelagic zone to tropical rainforest rivers. These fishes also exhibit a diversity of diets, including piscivory, herbivory, and zooplanktivory. We investigated how diet and habitat affected the molecular evolution of cone opsins, which play a key role in bright light and colour vision and are tightly linked to ecology and life history. We analyzed a targeted-capture dataset to reconstruct the evolutionary history of beloniforms and assemble cone opsin sequences. We implemented codon-based clade models of evolution to examine how molecular evolution was affected by habitat and diet. We found high levels of positive selection in medium- and long-wavelength beloniform opsins, with piscivores showing increased positive selection in medium-wavelength opsins and zooplanktivores showing increased positive selection in long-wavelength opsins. In contrast, short-wavelength opsins showed purifying selection. While marine/freshwater habitat transitions have an effect on opsin molecular evolution, we found that diet plays a more important role. Our study suggests that evolutionary transitions along ecological axes produce complex adaptive interactions that affect patterns of selection on genes that underlie vision.

Scaling up Functional Analyses of the G Protein-Coupled Receptor Rhodopsin.

Eukaryotic cells use G protein-coupled receptors (GPCRs) to convert external stimuli into internal signals to elicit cellular responses. However, how mutations in GPCR-coding genes affect GPCR activation and downstream signaling pathways remain poorly understood. Approaches such as deep mutational scanning show promise in investigations of GPCRs, but a high-throughput method to measure rhodopsin activation has yet to be achieved. Here, we scale up a fluorescent reporter assay in budding yeast that we engineered to study rhodopsin's light-activated signal transduction. Using this approach, we measured the mutational effects of over 1200 individual human rhodopsin mutants, generated by low-frequency random mutagenesis of the GPCR rhodopsin (RHO) gene. Analysis of the data in the context of rhodopsin's three-dimensional structure reveals that transmembrane helices are generally less tolerant to mutations compared to flanking helices that face the lipid bilayer, which suggest that mutational tolerance is contingent on both the local environment surrounding specific residues and the specific position of these residues in the protein structure. Comparison of functional scores from our screen to clinically identified rhodopsin disease variants found many pathogenic mutants to be loss of function. Lastly, functional scores from our assay were consistent with a complex counterion mechanism involved in ligand-binding and rhodopsin activation. Our results demonstrate that deep mutational scanning is possible for rhodopsin activation and can be an effective method for revealing properties of mutational tolerance that may be generalizable to other transmembrane proteins.

Adaptive Evolution of Nearctic Deepwater Fish Vision: Implications for Assessing Functional Variation for Conservation.

Intraspecific functional variation is critical for adaptation to rapidly changing environments. For visual opsins, functional variation can be characterized in vitro and often reflects a species' ecological niche but is rarely considered in the context of intraspecific variation or the impact of recent environmental changes on species of cultural or commercial significance. Investigation of adaptation in postglacial lakes can provide key insight into how rapid environmental changes impact functional evolution. Here, we report evidence for molecular adaptation in vision in 2 lineages of Nearctic fishes that are deep lake specialists: ciscoes and deepwater sculpin. We found depth-related variation in the dim-light visual pigment rhodopsin that evolved convergently in these 2 lineages. In vitro characterization of spectral sensitivity of the convergent deepwater rhodopsin alleles revealed blue-shifts compared with other more widely distributed alleles. These blue-shifted rhodopsin alleles were only observed in deep clear postglacial lakes with underwater visual environments enriched in blue light. This provides evidence of remarkably rapid and convergent visual adaptation and intraspecific functional variation in rhodopsin. Intraspecific functional variation has important implications for conservation, and these fishes are of conservation concern and great cultural, commercial, and nutritional importance to Indigenous communities. We collaborated with the Saugeen Ojibway Nation to develop and test a metabarcoding approach that we show is efficient and accurate in recovering the ecological distribution of functionally relevant variation in rhodopsin. Our approach bridges experimental analyses of protein function and genetics-based tools used in large-scale surveys to better understand the ecological extent of adaptive functional variation.

Evolution of a novel regulatory mechanism of hypoxia inducible factor in hypoxia-tolerant electric fishes.

Hypoxia is a significant source of metabolic stress that activates many cellular pathways involved in cellular differentiation, proliferation, and cell death. Hypoxia is also a major component in many human diseases and a known driver of many cancers. Despite the challenges posed by hypoxia, there are animals that display impressive capacity to withstand lethal levels of hypoxia for prolonged periods of time and thus offer a gateway to a more comprehensive understanding of the hypoxic response in vertebrates. The weakly electric fish genus Brachyhypopomus inhabits some of the most challenging aquatic ecosystems in the world, with some species experiencing seasonal anoxia, thus providing a unique system to study the cellular and molecular mechanisms of hypoxia tolerance. In this study, we use closely related species of Brachyhypopomus that display a range of hypoxia tolerances to probe for the underlying molecular mechanisms via hypoxia inducible factors (HIFs)-transcription factors known to coordinate the cellular response to hypoxia in vertebrates. We find that HIF1⍺ from hypoxia tolerant Brachyhypopomus species displays higher transactivation in response to hypoxia than that of intolerant species, when overexpressed in live cells. Moreover, we identified two SUMO-interacting motifs near the oxygen-dependent degradation and transactivation domains of the HIF1⍺ protein that appear to boost transactivation of HIF1, regardless of the genetic background. Together with computational analyses of selection, this shows that evolution of HIF1⍺ are likely to underlie adaptations to hypoxia tolerance in Brachyhypopomus electric fishes, with changes in two SUMO-interacting motifs facilitating the mechanism of this tolerance.

Convergent evolution of dim light vision in owls and deep-diving whales.

Animals with enhanced dim-light sensitivity are at higher risk of light-induced retinal degeneration when exposed to bright light conditions.1,2,3,4 This trade-off is mediated by the rod photoreceptor sensory protein, rhodopsin (RHO), and its toxic vitamin A chromophore by-product, all-trans retinal.5,6,7,8 Rod arrestin (Arr-1) binds to RHO and promotes sequestration of excess all-trans retinal,9,10 which has recently been suggested as a protective mechanism against photoreceptor cell death.2,11 We investigated Arr-1 evolution in animals at high risk of retinal damage due to periodic bright-light exposure of rod-dominated retinas. Here, we find the convergent evolution of enhanced Arr-1/RHO all-trans-retinal sequestration in owls and deep-diving whales. Statistical analyses reveal a parallel acceleration of Arr-1 evolutionary rates in these lineages, which is associated with the introduction of a rare Arr-1 mutation (Q69R) into the RHO-Arr-1 binding interface. Using in vitro assays, we find that this single mutation significantly enhances RHO-all-trans-retinal sequestration by ∼30%. This functional convergence across 300 million years of evolutionary divergence suggests that Arr-1 and RHO may play an underappreciated role in the photoprotection of the eye, with potentially vast clinical significance.

Adaptation of Antarctic Icefish Vision to Extreme Environments.

Extreme environments, such as Antarctic habitats, present major challenges for many biological processes. Antarctic icefishes (Crynotothenioidea) represent a compelling system to investigate the molecular basis of adaptation to cold temperatures. Here, we explore how the sub-zero habitats of Antarctic icefishes have impacted rhodopsin (RH1) function, the temperature-sensitive dim-light visual pigment found in rod photoreceptors. Using likelihood models and ancestral reconstruction, we find that accelerated evolutionary rates in icefish RH1 underlie unique amino acid mutations absent from other deep-dwelling fishes, introduced before (S160A) and during (V259M) the onset of modern polar conditions. Functional assays reveal that these mutations red-shift rhodopsin spectral absorbance, consistent with spectral irradiance under sea ice. These mutations also lower the activation energy associated with retinal release of the light-activated RH1, and accelerate its return to the dark state, likely compensating for a cold-induced decrease in kinetic rates. These are adaptations in key properties of rhodopsin that mediate rod sensitivity and visual performance in the cold dark seas of the Antarctic.

Ancient whale rhodopsin reconstructs dim-light vision over a major evolutionary transition: Implications for ancestral diving behavior.

Cetaceans are fully aquatic mammals that descended from terrestrial ancestors, an iconic evolutionary transition characterized by adaptations for underwater foraging via breath-hold diving. Although the evolutionary history of this specialized behavior is challenging to reconstruct, coevolving sensory systems may offer valuable clues. The dim-light visual pigment, rhodopsin, which initiates phototransduction in the rod photoreceptors of the eye, has provided insight into the visual ecology of depth in several aquatic vertebrate lineages. Here, we use ancestral sequence reconstruction and protein resurrection experiments to quantify light-activation metrics in rhodopsin pigments from ancestors bracketing the cetacean terrestrial-to-aquatic transition. By comparing multiple reconstruction methods on a broadly sampled cetartiodactyl species tree, we generated highly robust ancestral sequence estimates. Our experimental results provide direct support for a blue-shift in spectral sensitivity along the branch separating cetaceans from terrestrial relatives. This blue-shift was 14 nm, resulting in a deep-sea signature (λmax = 486 nm) similar to many mesopelagic-dwelling fish. We also discovered that the decay rates of light-activated rhodopsin increased in ancestral cetaceans, which may indicate an accelerated dark adaptation response typical of deeper-diving mammals. Because slow decay rates are thought to help sequester cytotoxic photoproducts, this surprising result could reflect an ecological trade-off between rod photoprotection and dark adaptation. Taken together, these ancestral shifts in rhodopsin function suggest that some of the first fully aquatic cetaceans could dive into the mesopelagic zone (>200 m). Moreover, our reconstructions indicate that this behavior arose before the divergence of toothed and baleen whales.

Simultaneous Expression of UV and Violet SWS1 Opsins Expands the Visual Palette in a Group of Freshwater Snakes.

Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and dN/dS ∼1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV-violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.

Uniform trichromacy in Alouatta caraya and Alouatta seniculus: behavioural and genetic colour vision evaluation.

Primate colour vision depends on a matrix of photoreceptors, a neuronal post receptoral structure and a combination of genes that culminate in different sensitivity through the visual spectrum. Along with a common cone opsin gene for short wavelengths (sws1), Neotropical primates (Platyrrhini) have only one cone opsin gene for medium-long wavelengths (mws/lws) per X chromosome while Paleotropical primates (Catarrhini), including humans, have two active genes. Therefore, while female platyrrhines may be trichromats, males are always dichromats. The genus Alouatta is inferred to be an exception to this rule, as electrophysiological, behavioural and molecular analyses indicated a potential for male trichromacy in this genus. However, it is very important to ascertain by a combination of genetic and behavioural analyses whether this potential translates in terms of colour discrimination capability. We evaluated two howler monkeys (Alouatta spp.), one male A. caraya and one female A. seniculus, using a combination of genetic analysis of the opsin gene sequences and a behavioral colour discrimination test not previously used in this genus. Both individuals completed the behavioural test with performances typical of trichromatic colour vision and the genetic analysis of the sws1, mws, and lws opsin genes revealed three different opsin sequences in both subjects. These results are consistent with uniform trichromacy in both male and female, with presumed spectral sensitivity peaks similar to Catarrhini, at ~ 430 nm, 532 nm, and 563 nm for S-, M- and L-cones, respectively.

Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.

Evolution, inactivation and loss of short wavelength-sensitive opsin genes during the diversification of Neotropical cichlids.

Natural variation in the number, expression and function of sensory genes in an organism's genome is often tightly linked to different ecological and evolutionary forces. Opsin genes, which code for the first step in visual transduction, are ideal models for testing how ecological factors such as light environment may influence visual system adaptation. Neotropical cichlid fishes are a highly ecologically diverse group that evolved in a variety of aquatic habitats, including black (stained), white (opaque) and clear waters. We used cross-species exon capture to sequence Neotropical cichlid short wavelength-sensitive (SWS) opsins, which mediate ultraviolet (UV) to blue visual sensitivity. Neotropical cichlid SWS1 opsin (UV-sensitive) underwent a relaxation of selective constraint during the early phases of cichlid diversification in South America, leading to pseudogenization and loss. Conversely, SWS2a (blue-sensitive) experienced a burst of episodic positive selection at the base of the South American cichlid radiation. This burst coincides with SWS1 relaxation and loss, and is consistent with findings in ecomorphological studies characterizing a period of extensive ecological divergence in Neotropical cichlids. We use ancestral sequence reconstruction and protein modelling to investigate mutations along this ancestral branch that probably modified SWS2a function. Together, our results suggest that variable light environments played a prominent early role in shaping SWS opsin diversity during the Neotropical cichlid radiation. Our results also illustrate that long-term evolution under light-limited conditions in South America may have reduced visual system plasticity; specifically, early losses of UV sensitivity may have constrained the evolutionary trajectory of Neotropical cichlid vision.

Recreated Ancestral Opsin Associated with Marine to Freshwater Croaker Invasion Reveals Kinetic and Spectral Adaptation.

Rhodopsin, the light-sensitive visual pigment expressed in rod photoreceptors, is specialized for vision in dim-light environments. Aquatic environments are particularly challenging for vision due to the spectrally dependent attenuation of light, which can differ greatly in marine and freshwater systems. Among fish lineages that have successfully colonized freshwater habitats from ancestrally marine environments, croakers are known as highly visual benthic predators. In this study, we isolate rhodopsins from a diversity of freshwater and marine croakers and find that strong positive selection in rhodopsin is associated with a marine to freshwater transition in South American croakers. In order to determine if this is accompanied by significant shifts in visual abilities, we resurrected ancestral rhodopsin sequences and tested the experimental properties of ancestral pigments bracketing this transition using in vitro spectroscopic assays. We found the ancestral freshwater croaker rhodopsin is redshifted relative to its marine ancestor, with mutations that recapitulate ancestral amino acid changes along this transitional branch resulting in faster kinetics that are likely to be associated with more rapid dark adaptation. This could be advantageous in freshwater due to the redshifted spectrum and relatively narrow interface and frequent transitions between bright and dim-light environments. This study is the first to experimentally demonstrate that positively selected substitutions in ancestral visual pigments alter protein function to freshwater visual environments following a transition from an ancestrally marine state and provides insight into the molecular mechanisms underlying some of the physiological changes associated with this major habitat transition.

Simultaneous expression of UV and violet SWS1 opsins expands the visual palette in a group of freshwater snakes

Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and dN/dS ∼1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV–violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.

Corticotropin-Releasing Factor: An Ancient Peptide Family Related to the Secretin Peptide Superfamily.

Corticotropin-releasing factor (CRF) is the hypothalamic releasing peptide that regulates the hypothalamic-pituitary-adrenal/inter-renal (HPA/I) axis in vertebrates. Over the last 25 years, there has been considerable discussion on its paralogs genes, urotensin-I/urocortin-1, and urocortins-2 and-3 and their subsequent role in the vertebrate stress response. Phylogenetically, the CRF family of peptides also belong to the diverse assemblage of Secretin- and Calcitonin-based peptides as evidenced by comparative-based studies of both their ligand and G-protein-coupled receptor (GPCR) structures. Despite this, the common origin of this large assemblage of peptides has not been ascertained. An unusual peptide, teneurin-C-terminal associated peptide (TCAP), reported in 2004, comprises the distal extracellular tip of the teneurin transmembrane proteins. Further studies indicated that this teneurin region binds to the latrophilin family of GPCRs. Initially thought to be a member of the Secretin GPCR family, evidence indicates that the latrophilins are a member of the Adhesion family of GPCRs and are related to the common ancestor of both Adhesion and Secretin GPCR families. In this study, we posit that TCAP may be a distantly related ancestor of the CRF-Calcitonin-Secretin peptide family and evolved near the base of metazoan phylogeny.

Convergent patterns of evolution of mitochondrial oxidative phosphorylation (OXPHOS) genes in electric fishes.

The ability to generate and detect electric fields has evolved in several groups of fishes as a means of communication, navigation and, occasionally, predation. The energetic burden required can account for up to 20% of electric fishes' daily energy expenditure. Despite this, molecular adaptations that enable electric fishes to meet the metabolic demands of bioelectrogenesis remain unknown. Here, we investigate the molecular evolution of the mitochondrial oxidative phosphorylation (OXPHOS) complexes in the two most diverse clades of weakly electric fishes-South American Gymnotiformes and African Mormyroidea, using codon-based likelihood approaches. Our analyses reveal that although mitochondrial OXPHOS genes are generally subject to strong purifying selection, this constraint is significantly reduced in electric compared to non-electric fishes, particularly for complexes IV and V. Moreover, analyses of concatenated mitochondrial genes show strong evidence for positive selection in complex I genes on the two branches associated with the independent evolutionary origins of electrogenesis. These results suggest that adaptive evolution of proton translocation in the OXPHOS cellular machinery may be associated with the evolution of bioelectrogenesis. Overall, we find striking evidence for remarkably similar effects of electrogenesis on the molecular evolution of mitochondrial OXPHOS genes in two independently derived clades of electrogenic fishes. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Screening of Chemical Libraries Using a Yeast Model of Retinal Disease.

Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9-cis retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z' factor of 0.65 and a signal-to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small-molecule libraries, as they are robust and cost-effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.

Evolutionary signatures of photoreceptor transmutation in geckos reveal potential adaptation and convergence with snakes.

Most vertebrates use a combination of rod and cone photoreceptors to enable vision in conditions ranging from starlight to direct sunlight. Nocturnal geckos, however, have simplex retinas that contain only rods in terms of morphology and physiology, but these rods are thought to be derived from cones through an evolutionary process known as photoreceptor transmutation. To investigate this, we generated eye transcriptomes and analyzed patterns of phototransduction gene evolution in geckos in comparison to other reptiles. We confirm that geckos have lost several major components of the rod phototransduction pathway, including rod opsin (RH1), which we identified as a pseudogene in multiple genomes. We also identified a partial rod transducin transcript, but found no evidence of the protein in retinal sections. However, we find that geckos express several complete rod phototransduction transcripts in the eye, which may contribute to the rod-like physiology of nocturnal gecko photoreceptors. Finally, we found surprising evidence that even though photoreceptor transmutation evolved independently in geckos and snakes, they have experienced parallel shifts in selective constraint on phototransduction genes. These results implicate adaptive change in the underlying molecular machinery of visual transduction, in addition to the convergent changes in cellular morphology, during photoreceptor transmutation.

To see or not to see: molecular evolution of the rhodopsin visual pigment in neotropical electric fishes.

Functional variation in rhodopsin, the dim-light-specialized visual pigment, frequently occurs in species inhabiting light-limited environments. Variation in visual function can arise through two processes: relaxation of selection or adaptive evolution improving photon detection in a given environment. Here, we investigate the molecular evolution of rhodopsin in Gymnotiformes, an order of mostly nocturnal South American fishes that evolved sophisticated electrosensory capabilities. Our initial sequencing revealed a mutation associated with visual disease in humans. As these fishes are thought to have poor vision, this would be consistent with a possible sensory trade-off between the visual system and a novel electrosensory system. To investigate this, we surveyed rhodopsin from 147 gymnotiform species, spanning the order, and analysed patterns of molecular evolution. In contrast with our expectation, we detected strong selective constraint in gymnotiform rhodopsin, with rates of non-synonymous to synonymous substitutions lower in gymnotiforms than in other vertebrate lineages. In addition, we found evidence for positive selection on the branch leading to gymnotiforms and on a branch leading to a clade of deep-channel specialized gymnotiform species. We also found evidence that deleterious effects of a human disease-associated substitution are likely to be masked by epistatic substitutions at nearby sites. Our results suggest that rhodopsin remains an important component of the gymnotiform sensory system alongside electrolocation, and that photosensitivity of rhodopsin is well adapted for vision in dim-light environments.