Biology subjects

Cortesi, F.

Publications and source records attributed to Cortesi, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Genomic structural variation in clownfish adaptive radiation

Clownfishes are a complex of 28 species that rapidly diversified after acquiring mutualism with host sea anemones around 15 million years ago. The genomic mechanisms behind this radiation are only beginning to emerge, and structural variants (SVs) have received little attention. Previous phylogenomic studies suggested two large inversions on chromosome 18, but their distribution across the clownfish phylogeny and their potential effects have not been formally explored. SVs can disrupt gene function and regulation or alter gene dosage, and they have been shown to play a central role in adaptive evolution and diversification across many taxa. Here, we characterized SVs across the clownfish radiation using long-read PacBio sequencing, generating high-quality genome assemblies for sixteen species spanning the main lineages of the genus. We identified and catalogued nearly 130,000 SVs spanning close to 570 Mb. Short insertions and deletions were the most numerous, but large inversions accounted for most of the affected sequence. No SV was consistently associated with host specialization. We nevertheless confirmed a single inversion of approximately 17 Mb on chromosome 18, clarifying earlier reports of two separate rearrangements at this locus, and identified an additional inversion on chromosome 9 whose distribution suggests a history of hybridization. This study provides the first genome-wide characterization of structural variation in clownfishes, revealing extensive SVs despite the group's rapid and recent radiation. It also delivers new, high-quality genomic resources that will support future research into this iconic group of coral reef fishes and their adaptation to sea anemones.

genomics↗

bioRxiv2026

Red light mitigates ALAN-induced memory impairment in the clownfish Amphiprion ocellaris

Artificial light at night (ALAN) is an increasingly pervasive anthropogenic pollutant in coastal ecosystems. However, its effects on brain function and behaviour in reef fishes remain poorly understood. Here, we investigated the impact of ALAN on learning and memory in the clownfish, Amphiprion ocellaris, a diurnal reef fish with well-characterised photoreceptive systems. Cognitive performance was assessed using a colour discrimination task following exposure to ALAN of varying intensities and spectral compositions. ALAN impaired learning and memory after only two consecutive nights of exposure, with effects emerging at intensities as low as 0.5 lux. At higher intensities (5-50 lux), memory was consistently disrupted under white-cool, white-warm, blue, and green LED light, whereas red light had no detectable effect. Together, these findings demonstrate that ALAN rapidly alters cognitive performance in clownfish and that these effects are strongly wavelength-dependent. Our results highlight the vulnerability of diurnal reef-associated fishes to nocturnal light pollution and underscore the need for ecologically informed lighting strategies that use longer-wavelength red-emitting light sources to reduce both the intensity and spectral impact of ALAN in coastal environments.

animal behavior and cognition↗

bioRxiv2024

Deep-sea fish reveal alternative pathway for vertebrate visual development

Vertebrate vision is accomplished by two phenotypically distinct types of photoreceptors in the retina: the saturation-resistant cones for the detection of bright light and the highly sensitive rods for dim light conditions [1]. The current dogma is that, during development, all vertebrates initially feature a cone-dominated retina, and rods are added later [2, 3]. By studying the ontogeny of vision in three species of deep-sea fishes, we show that their larvae express cone-specific genes in photoreceptors with rod-like morphologies. Through development, these fishes either retain this rod-like cone retina (Maurolicus mucronatus) or switch to a retina with true rod photoreceptors with expression of rod-specific genes and transcription factors (Vinciguerria mabahiss and Benthosema pterotum). In contrast to the larvae of most marine fishes, which inhabit the bright upper layer of the open ocean, the larvae of deep-sea fishes occur deeper, exposing them to a dimmer light environment [4-7]. Spectral maxima predictions from molecular dynamics simulations and environmental light estimations suggest that using transmuted photoreceptors that combine the characteristics of both cones and rods maximises visual performance in these dimmer light conditions. Our findings provide molecular, morphological, and functional evidence for the evolution of an alternative developmental pathway for vertebrate vision.

evolutionary biology↗

bioRxiv2024

Evolution of the non-visual and visual opsin gene repertoire in ray-finned fishes

Photoreception - the detection of light for image formation (vision) as well as for non-image-forming purposes (circadian regulation, DNA repair) - is critical to the survival of most animals. In vertebrates, photoreception is mediated by opsin proteins, which are classified, according to their function, into visual and non-visual opsins. Here, we provide the most comprehensive study to date on the evolution of the opsin gene family in the largest class of vertebrate, fishes, with a particular focus on the understudied non-visual opsins. Based on an in-depth analysis of 535 high-quality genomes, we document great variation in gene numbers in the different opsin gene subfamilies across ray-finned fishes and show that visual opsins are more prone to duplications and losses than non-visual opsins. We provide compelling evidence that visual and non-visual opsins co-evolve in ray-finned fishes, both in terms of copy numbers and selective pressures acting on their coding sequences, probably in response to the different photic environments they inhabit. Species that live in dim light or all in the dark (such as in caves or the deep sea) convergently reduced their visual and non-visual opsin gene repertoires, while polar species feature accelerated evolution in both. Fishes that rely on electroreception show a reduction in the number of visual and non-visual opsin genes and accelerated evolution of the remaining genes. We further found that genes of the phototransduction cascade co-evolve with opsins. Finally, that non-visual opsins are mainly expressed in testes and ovaries (next to the eyes) supports a function in gamete biology.

evolutionary biology↗

bioRxiv2024

Damsels in a hidden colour: development of ultraviolet sensitivity and colour patterns in damselfishes (Pomacentridae)

Damselfishes (Pomacentridae) are widespread and highly abundant on tropical coral reefs. They exhibit diverse body colouration within and between the [~]250 species and across ontogenetic stages. In addition to human visible colours (i.e., 400-700 nm), most adult damselfishes reflect ultraviolet (UV, 300-400 nm) colour patches. UV sensitivity and UV colour signals are essential for feeding and form the basis for a secret communication channel invisible to the many UV-blind predatory fish on the reef; however, how these traits develop across ontogenetic stages, and their distribution across the damselfish family is poorly characterised. Here, we used UV photography, phylogenetic reconstructions of opsin genes, differential gene expression analysis (DGE) of retinal samples, to investigate the development of UV vision and colour patterns in three ontogenetic stages (pre-settlement larval, juvenile, and adult) of eleven damselfish species. Using DGE, we found similar gene expression between juveniles and adults, which strongly differed from larvae. All species and all stages expressed at least one UV-sensitive sws1 opsin gene. However, UV body colour patterns only started to appear at the juvenile stage. Moreover, Pomacentrus species displayed highly complex UV body patterns that were correlated with the expression of two sws1 copies. This could mean that some damselfishes can discriminate colours that change only in their UV component. We demonstrate dramatic shifts in both UV sensitivity and UV colouration across the development stages of damselfish, while highlighting the importance of considering ontogeny when studying the coevolution of visual systems and colour signals.

ecology↗

bioRxiv2024

Specialization into Host Sea Anemones Impacted Clownfish Demographic Responses to Pleistocene Sea Level Changes

Fluctuating sea levels during the Pleistocene led to habitat loss and fragmentation, impacting the evolutionary trajectories of reef fishes. Species with specialized ecological requirements or habitat preferences, like clownfishes (Amphiprioninae), may have been particularly vulnerable due to their intricate dependence on sea anemones. The diverse host specializations within this group likely contributed distinct responses to sea-level fluctuations, differentially shaping their recent evolutionary histories. Leveraging a comprehensive genomic dataset, we reveal demographic patterns and connectivity dynamics across multiple populations of ten clownfish species under different host specializations. Host-generalist species demonstrated strong resilience to habitat perturbations, while those specialized on single hosts suffered dramatic bottlenecks linked to sea-level fluctuations. Spatial analyses revealed the significant role of oceanic currents in shaping clownfish genetic diversity landscapes. Dispersal barriers were driven by environmental variables, with the Coral Triangle emerging as a hub of genetic diversity. Our results reveal how clownfish associative behavior influences their population dynamics, holding major implications for their conservation such as the need to consider their mutualism with sea anemones, particularly on host-specialists, to ensure their survival in the face of climate threats. These findings extend broader principles of conservation, improving our understanding of species responses to ecological constraints and environmental changes over evolutionary timescales.

genomics↗

bioRxiv2024

Generation of the First Transgenic Line of the Iconic Coral Reef Fish Amphiprion ocellaris

The common clownfish, Amphiprion ocellaris, is an iconic coral reef fish, ubiquitous in the marine aquarium hobby and useful for studying a variety of biological processes (e.g., mutual symbiosis, ultraviolet vision, and protandrous sex change). Recently, CRISPR/Cas9 methods were developed for knocking out specific genes for mechanistic studies. Here, we expand the genetic toolkit for A. ocellaris by creating the first transgenic line using the Tol2 transposon system. Fertilized eggs were co-injected with Tol2 transposase mRNA and a plasmid encoding an Elongation factor 1 (Ef1): Green fluorescent protein (GFP) cassette at various concentrations, needle tip dimensions and timepoints post-fertilization. We compared various injection parameters and sterilization methods to maximize the survival of injected eggs. F0s (n=10) that were genotyped GFP+ were then raised to 6 months of age and crossed with wild-type (WT) females to confirm germline transmission. F1 offspring were also raised and crossed in the same manner. The highly efficient Tol2 transposon system resulted in a 37% rate of transgenesis for surviving eggs amounting to a 2.7% yield of all injected eggs surviving and being GFP+ (n= 160). Of these, 10 were raised to adulthood, 8 spawned, and 5/8 (62.5 %) produced GFP+ offspring. Further, two F1s crossed with WT females produced 53.8% and 54.2% GFP+ offspring respectively, confirming the creation of a stable line. This is, to our knowledge, the first generation of a transgenic line in any coral reef fish. The ability to express transgenes of interest in the iconic anemonefish opens the door to a new era of exploration into their fascinating biology.

bioengineering↗

bioRxiv2024

Eluding anemone nematocysts: are clownfish deprived of N-acetylated sugars in their skin mucus?

The clownfish - sea anemone system is a great example of symbiotic mutualism where host << toxicity >> does not impact its symbiont partner, although the underlying protection mechanism remains unclear. The regulation of nematocyst discharge in cnidarians involves N-acetylated sugars like sialic acid, that bind chemoreceptors on the tentacles of sea anemones, leading to the release of stings. It has been suggested that clownfish could be deprived of sialic acid on their skin surface, sparing them from being stung and facilitating mutualism with sea anemones. In this study, we sampled the skin mucus of two anemone symbionts, the clownfish Amphiprion akindynos and the juvenile damselfish Dascyllus trimaculatus, as well as two non-symbiotic adult damselfish Pomacentrus moluccensis and P. pavo. The free and total sialic acid content, including its conjugated form, and three other intermediates of this pathway were quantified using a stable isotope dilution mass spectrometry approach. We found significantly higher levels of sialic acid and its precursor in the non-symbiotic damselfishes. Concentrations of total sialic acid in anemone symbionts ranged between 13 {micro}M and 16 {micro}M, whereas the non-symbiotic damselfishes ranged between 21 {micro}M and 30 {micro}M. The presence of this metabolite and its precursors, as triggers of nematocyst discharge, in anemone symbionts, suggests that this is not the direct mechanism of protection or that the trigger is concentration dependent. This experiment demonstrates that anemone symbionts are not spared by nematocysts because of a lack of N-acetylated sugars, as previously thought.

molecular biology↗

bioRxiv2023

Recurrent gene flow events shaped the diversification of the clownfish skunk complex

Clownfish (subfamily Amphiprioninae) are an iconic group of coral reef fish that evolved a mutualistic interaction with sea anemones, which was shown to have triggered the adaptive radiation of the group. Within clownfishes, the skunk complex is particularly interesting as, besides ecological speciation, gene flow between species and hybrid speciation are suggested to have shaped the diversification of the group. We investigated, for the first time, the mechanisms underlying the diversification of this complex. By taking advantage of their disjunct geographical distribution, we obtained whole-genome data of sympatric and allopatric populations of the three main species of the complex (Amphiprion akallopisos, A. perideraion and A. sandaracinos). We examined the population structure, genomic divergence patterns and introgression signals, and performed demographic modeling to identify the most realistic diversification scenario. We excluded scenarios of strict isolation, of hybrid origin of A. sandaracinos, and ruled out the presence of extensive gene flow in sympatry. We discovered moderate gene flow from A. perideraion to the ancestor of A. akallopisos + A. sandaracinos and weak gene flow between the species in the Indo-Australian Archipelago throughout the diversification process of the group. We identified introgressed regions in A. sandaracinos and detected two large regions of high divergence in A. perideraion, likely maintained by the disruption of recombination. Altogether, our results show that ancestral hybridization events shaped the groups diversification. However, more recent gene flow is less pervasive than initially thought and suggests a role of host repartition or behavioral barriers in maintaining the genetic identity of the species in sympatry.

evolutionary biology↗

bioRxiv2023

Gene flow throughout the evolutionary history of a polymorphic and generalist clownfish

Even seemingly homogeneous on the surface, the oceans display high environmental heterogeneity across space and time. Indeed, different soft barriers structure the marine environment, which offers an appealing opportunity to study various evolutionary processes such as population differentiation and speciation. Here, we focus on Amphiprion clarkii (Actinopterygii; Perciformes), the most widespread of clownfishes that exhibits the highest colour polymorphism. Clownfishes can only disperse during a short pelagic larval phase before their sessile adult lifestyle, which might limit connectivity among populations, thus facilitating speciation events. Consequently, the taxonomic status of A. clarkii has been under debate. We used whole-genome resequencing data of 67 A. clarkii specimens spread across the Indian and Pacific Oceans to characterise the species population structure, demographic history, and colour polymorphism. We found that A. clarkii spread from the Indo-Pacific Ocean to the Pacific and Indian Oceans following a stepping-stone dispersal and that gene flow was pervasive throughout its demographic history. Moreover, edge populations exhibited more similar colouration patterns compared to central populations. However, we demonstrate that colour polymorphism is not associated with population structure, thus, colour phenotype is unreliable in assessing the taxonomic status of A. clarkii. Our study further highlights the power of whole-genome comparative studies to determine the taxonomy of geographically wide-ranging and phenotypically diverse species, supporting the status of A. clarkii as a single species.

evolutionary biology↗

bioRxiv2022

Anemonefish have finer color discrimination in the ultraviolet

In many animals, ultraviolet (UV) vision guides navigation, foraging, and communication, but few studies have addressed the contribution of UV vision to color discrimination, or behaviorally assessed UV discrimination thresholds. Here, we tested UV-color vision in an anemonefish (Amphiprion ocellaris) using a novel five-channel (RGB-V-UV) LED display designed to test UV perception. We first determined that the maximal sensitivity of the A. ocellaris UV cone was at [~]386 nm using microspectrophotometry. Three additional cone spectral sensitivities had maxima at [~]497, 515, and [~]535 nm, which together informed the modelling of the fishs color vision. Anemonefish behavioral discrimination thresholds for nine sets of colors were determined from their ability to distinguish a colored target pixel from grey distractor pixels of varying intensity. We found that A. ocellaris used all four cones to process color information and is therefore tetrachromatic, and fish were better at discriminating colors (i.e., color discrimination thresholds were lower, or more acute) when targets had UV chromatic contrast elicited by greater stimulation of the UV cone relative to other cone types. These findings imply that a UV component of color signals and cues improves their detectability, that likely increases the salience of anemonefish body patterns used in communication and the silhouette of zooplankton prey.

animal behavior and cognition↗

bioRxiv2022

Multiple rod layers increase the speed and sensitivity of vision in nocturnal reef fishes

Multibank retinas have rod photoreceptors stacked into multiple layers. They are found in many species of fish that inhabit dim environments and are one of the most common visual adaptations in the deep-sea. Despite its prevalence, the function of multibank retinas remained unknown. Two predominant theories, neither of which has been tested, have emerged: 1) they enhance sensitivity in dim light, and 2) they allow colour vision in dim light. To investigate the sensitivity hypothesis, we performed electrophysiological recordings and compared the rod pigments of three species of nocturnal reef fishes, two with a multibank retina (Neoniphon sammara and Myripristis violacea) and a control species with a single rod bank (Ostorhinchus compressus). Results indicated that nocturnal reef fishes with a multibank retina have higher temporal resolution of vision, as indicated by electrophysiology, and that their rhodopsin proteins likely also have faster retinal release kinetics, as suggested by amino acid substitutions. Electrophysiology also showed that the multibank retina conferred greater sensitivity to both dim and bright intensities than a single rod bank and this occurred at times when rod-derived signals usually dominate the visual response. This study provides the first functional evidence for enhanced dim-light sensitivity using a multibank retina while also suggesting novel roles for the adaptation in enhancing bright-light sensitivity and the speed of vision. SignificanceMost vertebrates have one layer of the dim-light active rod photoreceptors; however, some species have multiple layers, known as a multibank retina. We used electrophysiology on nocturnal reef fishes with and without multibank retinas to evaluate the sensory advantage of having multiple rod layers. We show that fish with multibank retinas have both faster vision and enhanced sensitivity to bright and dim light intensities. Thus, we resolve for the first time the function of multibank retinas - one of the most common visual adaptations in the deep sea. Our findings highlight an unconventional vertebrate visual system as well as the visual capabilities of fishes from the most vast (deep sea) and vibrant (reefs) ecosystems on the planet.

zoology↗

bioRxiv2022

Extensive hybridisation throughout clownfishes evolutionary history

The contribution of hybridisation in the generation of global species diversity has long been controversial among evolutionary biologists. However, it is now increasingly accepted that hybridisation has many impacts on the process of speciation. Notably, it is an important mechanism fostering adaptive radiation since it can generate new phenotypic combinations enabling the occupancy of new niches. Here, we focused on clownfish (Pomacentridae), a clade of 28 coral reef fishes displaying a mutualistic interaction with sea anemones. This behaviour is the key innovation that triggered adaptive radiation of clownfishes, as each species is able to occupy a different combination of host anemone species and habitat. Previous work suggested that hybridisation might be responsible for the extant diversity of clownfish species. To test this hypothesis, we analysed whole-genome datasets for each clownfish species. First, we reconstructed the phylogeny of the clade based on topology weighting methods, which enables the visualisation of the relationships between taxa across the genome. Then, we highlighted possible ancient hybridisation events based on a comparative genomic framework for detecting introgression in genomes. The resulting phylogeny is consistent with previous works based on a few mitochondrial and nuclear genes, and shallow nodes are now well supported in contrast to past studies. Furthermore, we detected multiple past hybridisation events throughout the evolutionary history of clownfishes, corroborating the potential role of hybridisation in the clownfish adaptive radiation. This study adds to the growing number of studies investigating the genomic mechanisms behind species diversification, drawing us closer to understanding how Earth biodiversity is generated.

evolutionary biology↗

bioRxiv2022

Developing and adult reef fish show rapid, reversible light-induced plasticity in their visual system

The visual capabilities of fish are optimised for their ecology and light environment over evolutionary time. Similarly, fish vision can adapt to regular changes in light conditions within their lifetime, e.g., ontogenetic or seasonal variation. However, we do not fully understand how vision responds to irregular short-term changes in the light environment, e.g., algal blooms and light pollution. In this study, we investigated the effect of short-term exposure to unnatural light conditions on opsin gene expression and retinal cell densities in larval and adult diurnal reef fish (convict surgeonfish; Acanthurus triostegus). Results revealed phenotypic plasticity in the retina across ontogeny, particularly in the larvae. The most substantial differences at both molecular and cellular levels were found under constant dim light, while constant bright light or simulated artificial light at night had a lesser effect. Under dim light, larvae and adults increased expression of the cone opsin genes, sws2a, rh2c and lws, within a few days and larvae also decreased densities of cones, inner nuclear layer cells and ganglion cells. These changes likely enhanced vision under the altered light conditions. Thus, our study suggests that plasticity mainly comes into play when conditions are extremely different to the species natural light environment, i.e., a diurnal fish in constant night. Finally, in a rescue experiment on adults, shifts in opsin expression were reverted within 24 hours. Overall, our study showed rapid, reversible light-induced changes in the retina of A. triostegus, demonstrating phenotypic plasticity in the visual system of a reef fish throughout life.

developmental biology↗

bioRxiv2022

Development of dim-light vision in the nocturnal coral reef fish family, Holocentridae

Developmental changes to the visual systems of animals are often associated with ecological shifts. Reef fishes experience a change in habitat between larval life in the shallow open ocean to juvenile and adult life on the reef. Some species also change their lifestyle over this period and become largely nocturnal. While these ecological transitions are well documented, little is known about the ontogeny of nocturnal reef fish vision. Here, we used histology and transcriptomics to investigate visual development in 12 representative species from both subfamilies, Holocentrinae (squirrelfishes) and Myripristinae (soldierfishes), in the nocturnal coral reef fish family, Holocentridae. Results revealed that the visual systems of holocentrids are initially well-adapted to photopic conditions with pre-settlement larvae having high cone densities, high cone opsin gene expression, a broad cone opsin gene repertoire (8 genes) and a multibank retina (i.e., stacked layers of rods) comprising up to two rod banks. At reef settlement, holocentrids started to invest more in their scotopic visual system and upregulated genes involved in cell differentiation/proliferation. By adulthood, they had well-developed scotopic vision with a rod-dominated multibank retina comprising 5-17 rod banks, increased summation of rods onto ganglion cells, high rod opsin gene expression, reduced cone opsin gene expression and repertoire (1-4 genes) and upregulated phototransduction genes. Finally, although the two subfamilies shared similar ecologies across development, their visual systems diverged after settlement, with Myripristinae investing more in scotopic vision than Holocentrinae. Hence, both ecology and phylogeny likely determine the development of the holocentrid visual system. Summary statementCoral reef fishes in the family Holocentridae remodel their retina at the cellular and molecular levels to adapt to a nocturnal lifestyle during development.

developmental biology↗

bioRxiv2021

Multiple ancestral and a plethora of recent gene duplications during the evolution of the green sensitive opsin genes (RH2) in teleost fishes

Vertebrates have four visual cone opsin classes that, together with a light-sensitive chromophore, provide sensitivity from the ultraviolet to the red wavelengths of light. The rhodopsin-like 2 (RH2) opsin is sensitive to the centre blue-green part of the spectrum, which is the most prevalent light underwater. While various vertebrate groups such as mammals and sharks have lost the RH2 gene, in teleost fishes this opsin has continued to proliferate. By investigating the genomes of 115 teleost species, we find that RH2 shows an extremely dynamic evolutionary history with repeated gene duplications, gene losses and gene conversion affecting entire orders, families and species. At least four ancestral duplications provided the substrate for todays RH2 diversity with duplications occurring in the common ancestors of Clupeocephala, Neoteleostei, and Acanthopterygii. Following these events, RH2 has continued to duplicate both in tandem and during lineage specific genome duplications. However, it has also been lost many times over so that in the genomes of extant teleosts, we find between zero to eight RH2 copies. Using retinal transcriptomes in a phylogenetic representative dataset of 30 species, we show that RH2 is expressed as the dominant green-sensitive opsin in almost all fish lineages. The exceptions are the Osteoglossomorpha (bony tongues and mooneyes) and several characin species that have lost RH2, and tarpons, other characins and gobies which do not or only lowly express the gene. These fishes instead express a green-shifted long-wavelength-sensitive LWS opsin. Our study highlights the strength of using modern genomic tools within a comparative framework to elucidate the detailed evolutionary history of gene families.

evolutionary biology↗

bioRxiv2021

Multiple ancestral duplications of the red-sensitive opsin gene (LWS) in teleost fishes and convergent spectral shifts to green vision in gobies

Photopigments, formed by an opsin protein bound to a light-sensitive chromophore, underlie vertebrate vision. Long-wavelength-sensitive (LWS) opsins mediate red-light detection, and most teleosts retain a single functional LWS1. A shorter-shifted green-sensitive paralog (LWS2) is found only in a few lineages. By mining teleost genomes and sequencing retinal transcriptomes, we identify elopomorphs as an additional lineage retaining LWS2 (alongside characins and osteoglossomorphs), and we reveal a previously overlooked shorter-shifted paralog, LWS3, restricted to gobies (Percomorpha) and arising from an ancient duplication. Structural modeling of twelve LWS opsins reveals convergent evolution at four key amino acid sites (214, 259, 261, 269) in the retinal-binding pocket, indicating convergent substitutions in human MWS, teleost LWS2, and goby LWS3 relative to red-sensitive counterparts, consistent with repeated spectral shifts toward green wavelengths. In several lineages--including characins, mormyrids, gobies, and primates--these shorter-shifted LWS opsins have functionally replaced the canonical green-sensitive RH2 opsin. Retinal transcriptomes and in situ hybridization demonstrate variable lws3 expression across gobies, with localization to a distinct double-cone member in Amblygobius phalaena, analogous to rh2 expression in other fish species. Together, these results show that repeated convergent evolution toward green sensitivity over 500 million years involves coordinated changes at the molecular, regulatory, and functional levels, providing a striking example of multilevel sensory adaptation. Significance statementVertebrate vision depends on opsins, which detect specific wavelengths of light. Most teleosts retain a single long-wavelength-sensitive opsin (LWS1), while a green-sensitive paralog (LWS2) occurs only in a few lineages. We identify a previously overlooked green-shifted opsin, LWS3, which is restricted to gobies, and show that LWS2 and LWS3 have repeatedly evolved green sensitivity through convergent amino acid changes. In parallel to the primate MWS/LWS evolution, these shorter-shifted opsins have functionally replaced the canonical green-sensitive RH2 opsin in multiple teleost lineages. By integrating genomic, structural, and expression data, we reveal how multilevel convergent evolution--from molecular tuning to photoreceptor specialization--has repeatedly shaped green-light vision over the past 500 million years of evolution, illustrating the remarkable flexibility of vertebrate visual systems.

evolutionary biology↗

bioRxiv2020

CRISPR/CAS9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutants

Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of eggs at the one-cell stage was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 50% of injected eggs. Further analysis of the subcloned mutant gene sequences revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in G0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful resource for future experimental studies that aim to elucidate gene function in anemonefishes and reef fishes in general.

molecular biology↗