Larval swimming in the sea anemone Nematostella vectensis is sensitive to a broad light spectrum and exhibits a wavelength-dependent behavioral switch.

In nearly all animals, light-sensing mediated by opsin visual pigments is important for survival and reproduction. Eyeless light-sensing systems, though vital for many animals, have received relatively less attention than forms with charismatic or complex eyes. Despite no single light-sensing organ, the sea anemone Nematostella vectensis has 29 opsin genes and multiple light-mediated behaviors throughout development and reproduction, suggesting a deceptively complex light-sensing system. To characterize one aspect of this light-sensing system, we analyzed larval swimming behavior at high wavelength resolution across the ultraviolet and visual spectrum. N. vectensis larvae respond to light at least from 315 to 650 nm, which is a broad sensitivity range even compared to many animals with complex eyes. Planktonic swimming is induced by ultraviolet (UV) and violet wavelengths until 420 nm. Between 420 and 430 nm a behavioral switch occurs where at wavelengths longer than 430 nm, larvae respond to light by swimming down. Swimming down toward the substrate is distinct from light avoidance, as animals do not exhibit positive or negative phototaxis at any wavelength tested. At wavelengths longer than 575 nm, animals in the water column take increasingly longer to respond and this behavior is more variable until 650 nm where larval response is no different from the dark, suggesting these longer wavelengths lie outside of their sensitivity range. Larval swimming is the only motile stage in the life history of N. vectensis, and increased planktonic swimming could lead to greater dispersal range in potentially damaging shallow environments with short-wavelength light exposure. Longer wavelength environments may indicate more suitable substrates for metamorphosis into the polyp stage, where the individual will remain for the rest of its life. Future work will test whether this robust behavior is mediated by multiple opsins.

Nematostella vectensis exemplifies the exceptional expansion and diversity of opsins in the eyeless Hexacorallia.

BACKGROUND: Opsins are the primary proteins responsible for light detection in animals. Cnidarians (jellyfish, sea anemones, corals) have diverse visual systems that have evolved in parallel with bilaterians (squid, flies, fish) for hundreds of millions of years. Medusozoans (e.g., jellyfish, hydroids) have evolved eyes multiple times, each time independently incorporating distinct opsin orthologs. Anthozoans (e.g., corals, sea anemones,) have diverse light-mediated behaviors and, despite being eyeless, exhibit more extensive opsin duplications than medusozoans. To better understand the evolution of photosensitivity in animals without eyes, we increased anthozoan representation in the phylogeny of animal opsins and investigated the large but poorly characterized opsin family in the sea anemone Nematostella vectensis. RESULTS: We analyzed genomic and transcriptomic data from 16 species of cnidarians to generate a large opsin phylogeny (708 sequences) with the largest sampling of anthozoan sequences to date. We identified 29 opsins from N. vectensis (NvOpsins) with high confidence, using transcriptomic and genomic datasets. We found that lineage-specific opsin duplications are common across Cnidaria, with anthozoan lineages exhibiting among the highest numbers of opsins in animals. To establish putative photosensory function of NvOpsins, we identified canonically conserved protein domains and amino acid sequences essential for opsin function in other animal species. We show high sequence diversity among NvOpsins at sites important for photoreception and transduction, suggesting potentially diverse functions. We further examined the spatiotemporal expression of NvOpsins and found both dynamic expression of opsins during embryonic development and sexually dimorphic opsin expression in adults. CONCLUSIONS: These data show that lineage-specific duplication and divergence has led to expansive diversity of opsins in eyeless cnidarians, suggesting opsins from these animals may exhibit novel biochemical functions. The variable expression patterns of opsins in N. vectensis suggest opsin gene duplications allowed for a radiation of unique sensory cell types with tissue- and stage-specific functions. This diffuse network of distinct sensory cell types could be an adaptive solution for varied sensory tasks experienced in distinct life history stages in Anthozoans.

Sex-linked gene traffic underlies the acquisition of sexually dimorphic UV color vision in Heliconius butterflies.

The acquisition of novel sexually dimorphic traits poses an evolutionary puzzle: How do new traits arise and become sex-limited? Recently acquired color vision, sexually dimorphic in animals like primates and butterflies, presents a compelling model for understanding how traits become sex-biased. For example, some Heliconius butterflies uniquely possess UV (ultraviolet) color vision, which correlates with the expression of two differentially tuned UV-sensitive rhodopsins, UVRh1 and UVRh2. To discover how such traits become sexually dimorphic, we studied Heliconius charithonia, which exhibits female-specific UVRh1 expression. We demonstrate that females, but not males, discriminate different UV wavelengths. Through whole-genome shotgun sequencing and assembly of the H. charithonia genome, we discovered that UVRh1 is present on the W chromosome, making it obligately female-specific. By knocking out UVRh1, we show that UVRh1 protein expression is absent in mutant female eye tissue, as in wild-type male eyes. A PCR survey of UVRh1 sex-linkage across the genus shows that species with female-specific UVRh1 expression lack UVRh1 gDNA in males. Thus, acquisition of sex linkage is sufficient to achieve female-specific expression of UVRh1, though this does not preclude other mechanisms, like cis-regulatory evolution from also contributing. Moreover, both this event, and mutations leading to differential UV opsin sensitivity, occurred early in the history of Heliconius. These results suggest a path for acquiring sexual dimorphism distinct from existing mechanistic models. We propose a model where gene traffic to heterosomes (the W or the Y) genetically partitions a trait by sex before a phenotype shifts (spectral tuning of UV sensitivity).

Cephalopod retinal development shows vertebrate-like mechanisms of neurogenesis.

Coleoid cephalopods, including squid, cuttlefish, and octopus, have large and complex nervous systems and high-acuity, camera-type eyes. These traits are comparable only to features that are independently evolved in the vertebrate lineage. The size of animal nervous systems and the diversity of their constituent cell types is a result of the tight regulation of cellular proliferation and differentiation in development. Changes in the process of development during evolution that result in a diversity of neural cell types and variable nervous system size are not well understood. Here, we have pioneered live-imaging techniques and performed functional interrogation to show that the squid Doryteuthis pealeii utilizes mechanisms during retinal neurogenesis that are hallmarks of vertebrate processes. We find that retinal progenitor cells in the squid undergo nuclear migration until they exit the cell cycle. We identify retinal organization corresponding to progenitor, post-mitotic, and differentiated cells. Finally, we find that Notch signaling may regulate both retinal cell cycle and cell fate. Given the convergent evolution of elaborate visual systems in cephalopods and vertebrates, these results reveal common mechanisms that underlie the growth of highly proliferative neurogenic primordia. This work highlights mechanisms that may alter ontogenetic allometry and contribute to the evolution of complexity and growth in animal nervous systems.

Insect opsins and evo-devo: what have we learned in 25 years?

The visual pigments known as opsins are the primary molecular basis for colour vision in animals. Insects are among the most diverse of animal groups and their visual systems reflect a variety of life histories. The study of insect opsins in the fruit fly Drosophila melanogaster has led to major advances in the fields of neuroscience, development and evolution. In the last 25 years, research in D. melanogaster has improved our understanding of opsin genotype-phenotype relationships while comparative work in other insects has expanded our understanding of the evolution of insect eyes via gene duplication, coexpression and homologue switching. Even so, until recently, technology and sampling have limited our understanding of the fundamental mechanisms that evolution uses to shape the diversity of insect eyes. With the advent of genome editing and in vitro expression assays, the study of insect opsins is poised to reveal new frontiers in evolutionary biology, visual neuroscience, and animal behaviour. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Multiple Mechanisms of Photoreceptor Spectral Tuning in Heliconius Butterflies.

The evolution of color vision is often studied through the lens of receptor gain relative to an ancestor with fewer spectral classes of photoreceptor. For instance, in Heliconius butterflies, a genus-specific UVRh opsin duplication led to the evolution of UV color discrimination in Heliconius erato females, a rare trait among butterflies. However, color vision evolution is not well understood in the context of loss. In Heliconius melpomene and Heliconius ismenius lineages, the UV2 receptor subtype has been lost, which limits female color vision in shorter wavelengths. Here, we compare the visual systems of butterflies that have either retained or lost the UV2 photoreceptor using intracellular recordings, ATAC-seq, and antibody staining. We identify several ways these butterflies modulate their color vision. In H. melpomene, chromatin reorganization has downregulated an otherwise intact UVRh2 gene, whereas in H. ismenius, pseudogenization has led to the truncation of UVRh2. In species that lack the UV2 receptor, the peak sensitivity of the remaining UV1 photoreceptor cell is shifted to longer wavelengths. Across Heliconius, we identify the widespread use of filtering pigments and co-expression of two opsins in the same photoreceptor cells. Multiple mechanisms of spectral tuning, including the molecular evolution of blue opsins, have led to the divergence of receptor sensitivities between species. The diversity of photoreceptor and ommatidial subtypes between species suggests that Heliconius visual systems are under varying selection pressures for color discrimination. Modulating the wavelengths of peak sensitivities of both the blue- and remaining UV-sensitive photoreceptor cells suggests that Heliconius species may have compensated for UV receptor loss.

Co-option of the limb patterning program in cephalopod eye development.

BACKGROUND: Across the Metazoa, similar genetic programs are found in the development of analogous, independently evolved, morphological features. The functional significance of this reuse and the underlying mechanisms of co-option remain unclear. Cephalopods have evolved a highly acute visual system with a cup-shaped retina and a novel refractive lens in the anterior, important for a number of sophisticated behaviors including predation, mating, and camouflage. Almost nothing is known about the molecular-genetics of lens development in the cephalopod. RESULTS: Here we identify the co-option of the canonical bilaterian limb patterning program during cephalopod lens development, a functionally unrelated structure. We show radial expression of transcription factors SP6-9/sp1, Dlx/dll, Pbx/exd, Meis/hth, and a Prdl homolog in the squid Doryteuthis pealeii, similar to expression required in Drosophila limb development. We assess the role of Wnt signaling in the cephalopod lens, a positive regulator in the developing Drosophila limb, and find the regulatory relationship reversed, with ectopic Wnt signaling leading to lens loss. CONCLUSION: This regulatory divergence suggests that duplication of SP6-9 in cephalopods may mediate the co-option of the limb patterning program. Thus, our study suggests that this program could perform a more universal developmental function in radial patterning and highlights how canonical genetic programs are repurposed in novel structures.

Krüppel-like factor/specificity protein evolution in the Spiralia and the implications for cephalopod visual system novelties.

The cephalopod visual system is an exquisite example of convergence in biological complexity. However, we have little understanding of the genetic and molecular mechanisms underpinning its elaboration. The generation of new genetic material is considered a significant contributor to the evolution of biological novelty. We sought to understand if this mechanism may be contributing to cephalopod-specific visual system novelties. Specifically, we identified duplications in the Krüppel-like factor/specificity protein (KLF/SP) sub-family of C2H2 zinc-finger transcription factors in the squid Doryteuthis pealeii. We cloned and analysed gene expression of the KLF/SP family, including two paralogs of the DpSP6-9 gene. These duplicates showed overlapping expression domains but one paralog showed unique expression in the developing squid lens, suggesting a neofunctionalization of DpSP6-9a. To better understand this neofunctionalization, we performed a thorough phylogenetic analysis of SP6-9 orthologues in the Spiralia. We find multiple duplications and losses of the SP6-9 gene throughout spiralian lineages and at least one cephalopod-specific duplication. This work supports the hypothesis that gene duplication and neofunctionalization contribute to novel traits like the cephalopod image-forming eye and to the diversity found within Spiralia.

Copy Number Variation and Expression Analysis Reveals a Nonorthologous Pinta Gene Family Member Involved in Butterfly Vision.

Vertebrate (cellular retinaldehyde-binding protein) and Drosophila (prolonged depolarization afterpotential is not apparent [PINTA]) proteins with a CRAL-TRIO domain transport retinal-based chromophores that bind to opsin proteins and are necessary for phototransduction. The CRAL-TRIO domain gene family is composed of genes that encode proteins with a common N-terminal structural domain. Although there is an expansion of this gene family in Lepidoptera, there is no lepidopteran ortholog of pinta. Further, the function of these genes in lepidopterans has not yet been established. Here, we explored the molecular evolution and expression of CRAL-TRIO domain genes in the butterfly Heliconius melpomene in order to identify a member of this gene family as a candidate chromophore transporter. We generated and searched a four tissue transcriptome and searched a reference genome for CRAL-TRIO domain genes. We expanded an insect CRAL-TRIO domain gene phylogeny to include H. melpomene and used 18 genomes from 4 subspecies to assess copy number variation. A transcriptome-wide differential expression analysis comparing four tissue types identified a CRAL-TRIO domain gene, Hme CTD31, upregulated in heads suggesting a potential role in vision for this CRAL-TRIO domain gene. RT-PCR and immunohistochemistry confirmed that Hme CTD31 and its protein product are expressed in the retina, specifically in primary and secondary pigment cells and in tracheal cells. Sequencing of eye protein extracts that fluoresce in the ultraviolet identified Hme CTD31 as a possible chromophore binding protein. Although we found several recent duplications and numerous copy number variants in CRAL-TRIO domain genes, we identified a single copy pinta paralog that likely binds the chromophore in butterflies.

Sexual Dimorphism and Retinal Mosaic Diversification following the Evolution of a Violet Receptor in Butterflies.

Numerous animal lineages have expanded and diversified the opsin-based photoreceptors in their eyes underlying color vision behavior. However, the selective pressures giving rise to new photoreceptors and their spectral tuning remain mostly obscure. Previously, we identified a violet receptor (UV2) that is the result of a UV opsin gene duplication specific to Heliconius butterflies. At the same time the violet receptor evolved, Heliconius evolved UV-yellow coloration on their wings, due to the pigment 3-hydroxykynurenine (3-OHK) and the nanostructure architecture of the scale cells. In order to better understand the selective pressures giving rise to the violet receptor, we characterized opsin expression patterns using immunostaining (14 species) and RNA-Seq (18 species), and reconstructed evolutionary histories of visual traits in five major lineages within Heliconius and one species from the genus Eueides. Opsin expression patterns are hyperdiverse within Heliconius. We identified six unique retinal mosaics and three distinct forms of sexual dimorphism based on ommatidial types within the genus Heliconius. Additionally, phylogenetic analysis revealed independent losses of opsin expression, pseudogenization events, and relaxation of selection on UVRh2 in one lineage. Despite this diversity, the newly evolved violet receptor is retained across most species and sexes surveyed. Discriminability modeling of behaviorally preferred 3-OHK yellow wing coloration suggests that the violet receptor may facilitate Heliconius color vision in the context of conspecific recognition. Our observations give insights into the selective pressures underlying the origins of new visual receptors.

Sexual dimorphism in the compound eye of Heliconius erato: a nymphalid butterfly with at least five spectral classes of photoreceptor.

Most butterfly families expand the number of spectrally distinct photoreceptors in their compound eye by opsin gene duplications together with lateral filter pigments; however, most nymphalid genera have limited diversity, with only three or four spectral types of photoreceptor. Here, we examined the spatial pattern of opsin expression and photoreceptor spectral sensitivities in Heliconius erato, a nymphalid with duplicate ultraviolet opsin genes, UVRh1 and UVRh2 We found that the H. erato compound eye is sexually dimorphic. Females express the two UV opsin proteins in separate photoreceptors, but males do not express UVRh1. Intracellular recordings confirmed that females have three short wavelength-sensitive photoreceptors (λmax=356, ∼390 and 470 nm), while males have two (λmax=390 and ∼470 nm). We also found two long wavelength-sensitive photoreceptors (green, λmax∼555 nm, and red, λmax∼600 nm), which express the same LW opsin. The red cell's shifted sensitivity is probably due to perirhabdomal filtering pigments. Sexual dimorphism of the UV-absorbing rhodopsins may reflect the females' need to discriminate conspecifics from co-mimics. Red-green color vision may be used to detect differences in red coloration on Heliconius wings, or for host-plant identification. Among nymphalids so far investigated, only H. erato is known to possess five spectral classes of photoreceptor; sexual dimorphism of the eye via suppression of one class of opsin (here UVRh1 in males) has not - to our knowledge - been reported in any animal.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings.

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Multiple recent co-options of Optix associated with novel traits in adaptive butterfly wing radiations.

BACKGROUND: While the ecological factors that drive phenotypic radiations are often well understood, less is known about the generative mechanisms that cause the emergence and subsequent diversification of novel features. Heliconius butterflies display an extraordinary diversity of wing patterns due in part to mimicry and sexual selection. Identifying the genetic drivers of this crucible of evolution is now within reach, as it was recently shown that cis-regulatory variation of the optix transcription factor explains red pattern differences in the adaptive radiations of the Heliconius melpomene and Heliconius erato species groups. RESULTS: Here, we compare the developmental expression of the Optix protein across a large phylogenetic sample of butterflies and infer that its color patterning role originated at the base of the neotropical passion-vine butterfly clade (Lepidoptera, Nymphalidae, Tribe: Heliconiini), shortly predating multiple Optix-driven wing pattern radiations in the speciose Heliconius and Eueides genera. We also characterize novel Optix and Doublesex expression in the male-specific pheromone wing scales of the basal heliconiines Dryas and Agraulis, thus illustrating that within the Heliconinii lineage, Optix has been evolutionarily redeployed in multiple contexts in association with diverse wing features. CONCLUSIONS: Our findings reveal that the repeated co-option of Optix into various aspects of wing scale specification was associated with multiple evolutionary novelties over a relatively short evolutionary time scale. In particular, the recruitment of Optix expression in colored scale cell precursors was a necessary condition to the explosive diversification of passion-vine butterfly wing patterns. The novel deployment of a gene followed by spatial modulation of its expression in a given cell type could be a common mode of developmental innovation for triggering phenotypic radiations.

Small molecule screen for compounds that affect vascular development in the zebrafish retina.

Blood vessel formation in the vertebrate eye is a precisely regulated process. In the human retina, both an excess and a deficiency of blood vessels may lead to a loss of vision. To gain insight into the molecular basis of vessel formation in the vertebrate retina and to develop pharmacological means of manipulating this process in a living organism, we further characterized the embryonic zebrafish eye vasculature, and performed a small molecule screen for compounds that affect blood vessel morphogenesis. The screening of approximately 2000 compounds revealed four small molecules that at specific concentrations affect retinal vessel morphology but do not produce obvious changes in trunk vessels, or in the neuronal architecture of the retina. Of these, two induce a pronounced widening of vessel diameter without a substantial loss of vessel number, one compound produces a loss of retinal blood vessels accompanied by a mild increase of their diameter, and finally one other generates a severe loss of retinal vessels. This work demonstrates the utility of zebrafish as a screening tool for small molecules that affect eye vasculature and presents several compounds of potential therapeutic importance.