Molecular Evidence that Only Two Opsin Subfamilies,the Blue Light- (SWS2) and Green Light-Sensitive (RH2), Drive Color Vision in Atlantic Cod (Gadus morhua)

Teleosts show a great variety in visual opsin complement, due to both gene duplication and gene loss. The repertoire ranges from one subfamily of visual opsins (scotopic vision) including rod opsin only retinas seen in many deep-sea species to multiple subfamilies of visual opsins in some pelagic species. We have investigated the opsin repertoire of Atlantic cod (Gadus morhua) using information in the recently sequenced cod genome and found that despite cod not being a deep sea species it lacks visual subfamilies sensitive towards the most extreme parts of the light spectra representing UV and red light. Furthermore, we find that Atlantic cod has duplicated paralogs of both blue-sensitive SWS2 and green-sensitive RH2 subfamilies, with members belonging to each subfamily linked in tandem within the genome (two SWS2-, and three RH2A genes, respectively). The presence of multiple cone opsin genes indicates that there have been duplication events in the cod ancestor SWS2 and RH2 opsins producing paralogs that have been retained in Atlantic. Our results are supported by expressional analysis of cone opsins, which further revealed an ontogenetic change in the array of cone opsins expressed. These findings suggest life stage specific programs for opsin regulation which could be linked to habitat changes and available light as the larvae is transformed into an early juvenile. Altogether we provide the first molecular evidence for color vision driven by only two families of cone opsins due to gene loss in a teleost.     

Cnidocyte discharge is regulated by light and opsin-mediated phototransduction

Background

Cnidocytes, the eponymous cell type of the Cnidaria, facilitate both sensory and secretory functions and are among the most complex animal cell types known. In addition to their structural complexity, cnidocytes display complex sensory attributes, integrating both chemical and mechanical cues from the environment into their discharge behavior. Despite more than a century of work aimed at understanding the sensory biology of cnidocytes, the specific sensory receptor genes that regulate their function remain unknown.

Results

Here we report that light also regulates cnidocyte function. We show that non-cnidocyte neurons located in battery complexes of the freshwater polyp Hydra magnipapillata specifically express opsin, cyclic nucleotide gated (CNG) ion channel and arrestin, which are all known components of bilaterian phototransduction cascades. We infer from behavioral trials that different light intensities elicit significant effects on cnidocyte discharge propensity. Harpoon-like stenotele cnidocytes show a pronounced diminution of discharge behavior under bright light conditions as compared to dim light. Further, we show that suppression of firing by bright light is ablated by cis-diltiazem, a specific inhibitor of CNG ion channels.

Conclusions

Our results implicate an ancient opsin-mediated phototransduction pathway and a previously unknown layer of sensory complexity in the control of cnidocyte discharge. These findings also suggest a molecular mechanism for the regulation of other cnidarian behaviors that involve both photosensitivity and cnidocyte function, including diurnal feeding repertoires and/or substrate-based locomotion. More broadly, our findings highlight one novel, non-visual function for opsin-mediated phototransduction in a cnidarian, the origins of which might have preceded the evolution of cnidarian eyes.     

Eye evolution: a question of genetic promiscuity

Animal eyes have long served as a classical example of independent origin followed by convergence of structures onto a few different solutions. During the past decade this view has been challenged by the discovery of shared developmental regulatory genes. The Pax6 gene in particular is almost universally employed for eye formation in bilaterian animals, despite widely different embryological origins. The resulting controversy on the multiple or single origins of animal eyes has gradually been sharpened by continuing discoveries of further general similarities in the genetic regulatory circuits of eye development. Recent work on gene expression in specified cell types, together with comparative studies of developmental genes in cnidarians, now show some promise to a solution of the controversy.     

Immunohistochemical evidence for multiple photosystems in box jellyfish

Cubomedusae (box jellyfish) possess a remarkable visual system with 24 eyes distributed in four sensory structures termed rhopalia. Each rhopalium is equipped with six eyes: two pairs of pigment cup eyes and two unpaired lens eyes. Each eye type probably captures specific features of the visual environment. To investigate whether multiple types of photoreceptor cells are present in the rhopalium, and whether the different eye types possess different types of photoreceptors, we have used immunohistochemistry with a range of vertebrate opsin antibodies to label the photoreceptors, and electroretinograms (ERG) to determine their spectral sensitivity. All photoreceptor cells of the two lens eyes of the box jellyfish Tripedalia cystophora and Carybdea marsupialis displayed immunoreactivity for an antibody directed against the zebrafish ultraviolet (UV) opsin, but not against any of eight other rhodopsin or cone opsin antibodies tested. In neither of the two species were the pigment cup eyes immunoreactive for any of the opsin antibodies. ERG analysis of the Carybdea lower lens eyes demonstrated a single spectral sensitivity maximum at 485 nm suggesting the presence of a single opsin type. Our data demonstrate that the lens eyes of box jellyfish utilize a single opsin and are thus color-blind, and that there is probably a different photopigment in the pigment cup eyes. The results support our hypothesis that the lens eyes and the pigment cup eyes of box jellyfish are involved in different and specific visual tasks.     

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]     

The opsins

The photosensitive molecule rhodopsin and its relatives consist of a protein moiety - an opsin - and a non-protein moiety - the chromophore retinal. Opsins, which are G-protein-coupled receptors (GPCRs), are found in animals, and more than a thousand have been identified so far. Detailed molecular phylogenetic analyses show that the opsin family is divided into seven subfamilies, which correspond well to functional classifications within the family: the vertebrate visual (transducin-coupled) and non-visual opsin subfamily, the encephalopsin/tmt-opsin subfamily, the G_q-coupled opsin/melanopsin subfamily, the G_o-coupled opsin subfamily, the neuropsin subfamily, the peropsin subfamily and the retinal photoisomerase subfamily. The subfamilies diversified before the deuterostomes (including vertebrates) split from the protostomes (most invertebrates), suggesting that a common animal ancestor had multiple opsin genes. Opsins have a seven-transmembrane structure similar to that of other GPCRs, but are distinguished by a lysine residue that is a retinal-binding site in the seventh helix. Accumulated evidence suggests that most opsins act as pigments that activate G proteins in a light-dependent manner in both visual and non-visual systems, whereas a few serve as retinal photoisomerases, generating the chromophore used by other opsins, and some opsins have unknown functions.     

The Expression of Three Opsin Genes from the Compound Eye of Helicoverpa armigera (Lepidoptera: Noctuidae) Is Regulated by a Circadian Clock,Light Conditions and Nutritional Status

Visual genes may become inactive in species that inhabit poor light environments, and the function and regulation of opsin components in nocturnal moths are interesting topics. In this study, we cloned the ultraviolet (UV), blue (BL) and long-wavelength-sensitive (LW) opsin genes from the compound eye of the cotton bollworm and then measured their mRNA levels using quantitative real-time PCR. The mRNA levels fluctuated over a daily cycle, which might be an adaptation of a nocturnal lifestyle, and were dependent on a circadian clock. Cycling of opsin mRNA levels was disturbed by constant light or constant darkness, and the UV opsin gene was up-regulated after light exposure. Furthermore, the opsin genes tended to be down-regulated upon starvation. Thus, this study illustrates that opsin gene expression is determined by multiple endogenous and exogenous factors and is adapted to the need for nocturnal vision, suggesting that color vision may play an important role in the sensory ecology of nocturnal moths.     

Genetic network of the eye in Platyhelminthes: expression and functional analysis of some players during planarian regeneration

Planarians are the free-living members (order Tricladida) of the phylum Platyhelminthes. They are triploblastic, acoelomate, unsegmented and located at the base of the Lophotrochozoa clade. Besides their huge regenerative capacity, planarians have simple eyes, considered similar to the prototypic eye suggested by Charles Darwin in his book 'On the Origin of Species'. The conserved genetic network that determines the initial steps of eye development across metazoans supports a monophyletic origin of the various eye types present in the animal kingdom. Here we summarise the pattern of expression of certain genes involved in the eye network that have been isolated in planarians, such as Otx, Pax-6, Six, Rax and opsin. We describe the effects of RNA interference-mediated loss of function on eye regeneration. Finally, we discuss the relevance of these findings for the evolution of the eye gene network.     

Opsins and cell fate in the Drosophila Bolwig organ: tricky lessons in homology inference

The Drosophila Bolwig organs are small photoreceptor bundles that facilitate the phototactic behavior of the larva. Comparative literature suggests that these highly reduced visual organs share evolutionary ancestry with the adult compound eye. A recent molecular genetic study produced the first detailed account of the mechanisms controlling differential opsin expression and photoreceptor subtype determination in these enigmatic eyes of the Drosophila larva. Here, the evolutionary implications are examined, taking into account the dynamic diversification of opsin genes and the spatial regulation of opsin homolog expression in other insects. It is concluded that, consistent with their common evolutionary roots, the Drosophila larval and adult eyes use the same mechanisms for the regulation of opsin expression and photoreceptor cell fate specification. Strikingly, the structurally highly derived Bolwig organs retained a more ancestral state of opsin expression and regulation. Inconspicuous in size, the Drosophila larval eyes deliver useful lessons in the reconstruction of homology between neuronal cell types with gene expression data, and on the conservative nature of gene regulatory network evolution during the emergence of novel organs from ancestral templates.     

Parallel and Convergent Evolution of the Dim-Light Vision Gene RH1 in Bats (Order: Chiroptera)

Rhodopsin, encoded by the gene Rhodopsin (RH1), is extremely sensitive to light, and is responsible for dim-light vision. Bats are nocturnal mammals that inhabit poor light environments. Megabats (Old-World fruit bats) generally have well-developed eyes, while microbats (insectivorous bats) have developed echolocation and in general their eyes were degraded, however, dramatic differences in the eyes, and their reliance on vision, exist in this group. In this study, we examined the rod opsin gene (RH1), and compared its evolution to that of two cone opsin genes (SWS1 and M/LWS). While phylogenetic reconstruction with the cone opsin genes SWS1 and M/LWS generated a species tree in accord with expectations, the RH1 gene tree united Pteropodidae (Old-World fruit bats) and Yangochiroptera, with very high bootstrap values, suggesting the possibility of convergent evolution. The hypothesis of convergent evolution was further supported when nonsynonymous sites or amino acid sequences were used to construct phylogenies. Reconstructed RH1 sequences at internal nodes of the bat species phylogeny showed that: (1) Old-World fruit bats share an amino acid change (S270G) with the tomb bat; (2) Miniopterus share two amino acid changes (V104I, M183L) with Rhinolophoidea; (3) the amino acid replacement I123V occurred independently on four branches, and the replacements L99M, L266V and I286V occurred each on two branches. The multiple parallel amino acid replacements that occurred in the evolution of bat RH1 suggest the possibility of multiple convergences of their ecological specialization (i.e., various photic environments) during adaptation for the nocturnal lifestyle, and suggest that further attention is needed on the study of the ecology and behavior of bats.     

Habitat light sets the boundaries for the rapid evolution of cichlid fish vision,while sexual selection can tune it within those limits

Cichlid fishes' famous diversity in body coloration is accompanied by a highly diverse and complex visual system. Although cichlids possess an unusually high number of seven cone opsin genes, they express only a subset of these during their ontogeny, accounting for their astonishing interspecific variation in visual sensitivities. Much of this diversity is thought to have been shaped by natural selection as cichlids inhabit a variety of habitats with distinct light environments. Also, sexual selection might have contributed to the observed visual diversity, and sexual dimorphism in coloration potentially co‐evolved with sexual dimorphism in opsin expression. We investigated sex‐specific opsin expression of several cichlids from Africa and the Neotropics and collected and integrated data sets on sex‐specific body coloration, species‐specific visual sensitivities, lens transmission and habitat light properties for some of them. We comparatively analysed this wide range of molecular and ecological data, illustrating how integrative approaches can address specific questions on the factors and mechanisms driving diversification, and the evolution of cichlid vision in particular. We found that both sexes expressed opsins at the same levels—even in sexually dimorphic cichlid species—which argues against coevolution of sexual dichromatism and differences in sex‐specific visual sensitivity. Rather, a combination of environmental light properties and body coloration shaped the diversity in spectral sensitivities among cichlids. We conclude that although cichlids are particularly colourful and diverse and often sexually dimorphic, it would appear that natural rather than sexual selection is a more powerful force driving visual diversity in this hyperdiverse lineage.     

Phylogenomics of trophically diverse cichlids disentangles processes driving adaptive radiation and repeated trophic transitions

Cichlid fishes of the tribe Tropheini are a striking case of adaptive radiation, exemplifying multiple trophic transitions between herbivory and carnivory occurring in sympatry with other established cichlid lineages. Tropheini evolved highly specialized eco‐morphologies to exploit similar trophic niches in different ways repeatedly and rapidly. To better understand the evolutionary history and trophic adaptations of this lineage, we generated a dataset of 532 targeted loci from 21 out of the 22 described Tropheini species. We resolved the Tropheini into seven monophyletic genera and discovered one to be polyphyletic. The polyphyletic genus, Petrochromis, represents three convergent origins of the algae grazing trophic specialization. This repeated evolution of grazing may have been facilitated by adaptive introgression as we found evidence for gene flow among algae grazing genera. We also found evidence of gene flow among algae browsing genera, but gene flow was restricted between herbivorous and carnivorous genera. Furthermore, we observed no evidence supporting a hybrid origin of this radiation. Our molecular evolutionary analyses suggest that opsin genes likely evolved in response to selection pressures associated with trophic ecology in the Tropheini. We found surprisingly little evidence of positive selection in coding regions of jaw‐shaping genes in this trophically diverse lineage. This suggests low degrees of freedom for further change in these genes, and possibly a larger role for regulatory variation in driving jaw adaptations. Our study emphasizes Tropheini cichlids as an important model for studying the evolution of trophic specialization and its role in speciation.     

Opsin evolution and expression in Arthropod compound Eyes and Ocelli: Insights from the cricket Gryllus bimaculatus

ABSTRACT: BACKGROUND: Opsins are key proteins in animal photoreception. Together with a light-sensitive group, the chromophore, they form visual pigments which initiate the visual transduction cascade when photoactivated. The spectral absorption properties of visual pigments are mainly determined by their opsins, and thus opsins are crucial to understand the adaptations of animal eyes. Studies on the phylogeny and expression pattern of opsins have received considerable attention, but our knowledge about insect visual opsins is still limited. Up to now, researchers have focused on holometabolous insects, while general conclusions require sampling from a broader range of taxa. We have therefore investigated visual opsins in the ocelli and compound eyes of the two-spotted cricket Gryllus bimaculatus, a hemimetabolous insect. RESULTS: Phylogenetic analyses place all identified cricket sequences within the three main visual opsin clades of insects. We assign three of these opsins to visual pigments found in the compound eyes with peak absorbances in the green (515 nm), blue (445 nm) and UV (332 nm) spectral range. Their expression pattern divides the retina into distinct regions: (1) the polarization-sensitive dorsal rim area with blue- and UV-opsin, (2) a newly-discovered ventral band of ommatidia with blue- and green-opsin and (3) the remainder of the compound eye with UV- and green-opsin. In addition, we provide evidence for two ocellar photopigments with peak absorbances in the green (511 nm) and UV (350 nm) spectral range, and with opsins that differ from those expressed in the compound eyes. CONCLUSIONS: Our data show that cricket eyes are spectrally more specialized than has previously been assumed, suggesting that similar adaptations in other insect species might have been overlooked. The arrangement of spectral receptor types within some ommatidia of the cricket compound eyes differs from the generally accepted pattern found in holometabolous insect taxa and awaits a functional explanation. From the opsin phylogeny, we conclude that gene duplications, which permitted differential opsin expression in insect ocelli and compound eyes, occurred independently in several insect lineages and are recent compared to the origin of the eyes themselves.     

Replication origins in metazoan chromosomes: fact or fiction?

The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition complex) next to an easily unwound DNA sequence at which replication can begin. The origin recognition complex provides a platform on which additional proteins assemble to form a pre-replication complex that can be activated at S-phase by specific protein kinases. Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication. Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial. In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone. Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification. Such complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development.