Many animals including birds, reptiles, insects, and teleost fishes can see ultraviolet (UV) light (shorter than 400 nm), which has functional importance for foraging and communication. For coral reef fishes, shallow reef environments transmit a broad spectrum of light, rich in UV, driving the evolution of diverse spectral sensitivities. However, the identities and sites of the specific visual genes that underly vision in reef fishes remain elusive and are useful in determining how evolution has tuned vision to suit life on the reef. We investigated the visual systems of 11 anemonefish (Amphiprioninae) species, specifically probing for the molecular pathways that facilitate UV-sensitivity. Searching the genomes of anemonefishes, we identified a total of eight functional opsin genes from all five vertebrate visual opsin subfamilies. We found rare instances of teleost UV-sensitive SWS1 opsin gene duplications, that produced two functionally-coding paralogs (SWS1α and SWS1β) and a pseudogene. We also found separate green sensitive RH2A opsin gene duplicates not yet reported in the family Pomacentridae. Transcriptome analysis revealed false clown anemonefish (Amphiprion ocellaris) expressed one rod opsin (RH1) and six cone opsins (SWS1β, SWS2B, RH2B, RH2A-1, RH2A-2, LWS) in the retina. Fluorescent in-situ hybridisation highlighted the (co-)expression of SWS1β with SWS2B in single cones, and either RH2B, RH2A, or RH2A together with LWS in different members of double cone photoreceptors (two single cones fused together). Our study provides the first in-depth characterisation of visual opsin genes found in anemonefishes and provides a useful basis for the further study of UV-vision in reef fishes.

Achromatic (luminance) vision is used by animals to perceive motion, pattern, space and texture. Luminance contrast sensitivity thresholds are often poorly characterised for individual species and are applied across a diverse range of perceptual contexts using over-simplified assumptions of an animal's visual system. Such thresholds are often estimated using the Receptor Noise Limited model (RNL) using quantum catch values and estimated noise levels of photoreceptors. However, the suitability of the RNL model to describe luminance contrast perception remains poorly tested.Here, we investigated context-dependent luminance discrimination using triggerfish (Rhinecanthus aculeatus) presented with large achromatic stimuli (spots) against uniform achromatic backgrounds of varying absolute and relative contrasts. ‘Dark’ and ‘bright’ spots were presented against relatively dark and bright backgrounds. We found significant differences in luminance discrimination thresholds across treatments. When measured using Michelson contrast, thresholds for bright spots on a bright background were significantly higher than for other scenarios, and the lowest threshold was found when dark spots were presented on dark backgrounds. Thresholds expressed in Weber contrast revealed increased contrast sensitivity for stimuli darker than their backgrounds, which is consistent with the literature. The RNL model was unable to estimate threshold scaling across scenarios as predicted by the Weber-Fechner law, highlighting limitations in the current use of the RNL model to quantify luminance contrast perception. Our study confirms that luminance contrast discrimination thresholds are context-dependent and should therefore be interpreted with caution.

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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 zygotes 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 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 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 starting point to further explore the potential application of CRISPR/Cas9 in A. ocellaris, as a platform for studying gene function in anemonefishes and other reef fishes.

1. The ability to see ultraviolet (UV) light (<400 nm) may have importance for foraging, communication or navigation in many taxa including insects, crustaceans, fishes, amphibians, reptiles, birds and mammals. Behavioural experiments reveal how vision mediates such behaviour; however, our knowledge of UV perception is constrained by the challenge of creating and calibrating stimuli that reflect or emit UV. Commonly used technologies for displaying visual stimuli-such as computer screens and printers-are designed for human vision and thus are unsuitable for testing UV perception. 2. To overcome this limitation, we designed and constructed a display with five spectral channels with peak wavelengths as follows: red at 629 nm, green at 526 nm, blue at 466 nm, violet at 395 nm and UV at 367 nm. Each pixel of the display consists of five LEDs with a baffle to prevent crosstalk from adjacent pixels and a diffuser to promote uniform colour mixing. The LEDs are driven by high-performance pulse-width-modulated constant-current drivers with a maximum flicker rate of 64 kHz and a maximum frame rate of 6.5 kHz. This method allows colour mixing with wavelengths as low as 350 nm to be calibrated and tested rapidly and concurrently. 3. To demonstrate the utility of this display, we conducted colour detection tests using the anemonefish, Amphiprion ocellaris, a species known to have UV-sensitive cones. Fish were able to associate pecking all target colours ('Blue', 'UV-grey' and 'UV') with a food reward, demonstrating for the first time, UV perception in A. ocellaris. 4. The RGB-V-UV LED display is a useful device for behavioural tests of colour vision across a broad spectrum (350-650 nm) visible to many animals and can be used to investigate various questions concerning animal perception, including colour discrimination and categorisation. We include design documents and source code so this system can be further developed and modified to investigate other visual behaviours in a variety of taxa. K E Y W O R D S animal behaviour, colour vision, coral reefs, ultraviolet vision, visual ecology This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.