Evolutionary adaptations to life on land include changes to an animal's physiology, morphology and behaviour. The visual systems of amphibious fishes show pronounced morphological adaptations; however, whether molecular changes also occur remains largely unknown. Here, we investigated the molecular evolution of visual opsin genes in blennies (Blenniidae), with a primary focus on the amphibious and terrestrial Salariini. Using retinal transcriptomes and amino acid comparisons in nine species from fully aquatic to terrestrial, we found limited sequence changes and no correlation between habitat and cone opsin gene expression. The 'red-sensitive' lws, the 'green-sensitive' rh2a, and two 'blue-sensitive' sws2aα and sws2aβ paralogues were expressed in all species, with the latter two showing pronounced phylogenetic inertia. Long-wavelength-dominated vision is likely beneficial for feeding on algae and detritus, the primary food source of most study species, and may be co-adapted to perceive red-coloured displays in terrestrial blennies. Conversely, a lack of 'ultraviolet-sensitive' sws1 expression coincides with ultraviolet-absorbing lenses in blennies, which likely evolved to protect the retina from the damaging effects of short-wavelength radiation, independent of habitat. Our data suggest that, at the molecular level, the visual systems that evolved in aquatic blennies have been retained in species that have progressively transitioned onto land.

Colour vision arises from the comparison of spectral input across at least two differently tuned photoreceptors. In most vertebrates, colour discrimination is mediated by two to four differently tuned cone photoreceptors, providing sensitivity from ultraviolet (< 400 nm) to red (∼600-700 nm) wavelengths. However, microspectrophotometry (MSP) indicated that the white-spotted boxfish, Ostracion meleagris, collected from Hawaiian reefs could possess five spectrally distinct cones. To explore the potential for increased cone diversity, we used a multidisciplinary approach to investigate colour vision in O. meleagris and its sister species, O. cubicus, from the Great Barrier Reef. Contrary to the MSP data, retinal transcriptomics and fluorescence in situ hybridisation found no evidence for five spectral sensitivities in either species. Instead, single cones (PR3) expressed sws2b, while the two members of the double cones (PR1 and PR2) expressed rh2a and rh2c opsins, respectively. Amino-acid modelling revealed putative visual pigments with uniformly spaced peak spectral sensitivities at 420 nm (SWS2B-based), 461-474 nm (RH2C) and 523-528 nm (RH2A). We compared the colour vision performance of O. cubicus with that of a well-studied trichromatic reef fish, the Picasso triggerfish (Rhinecanthus aculeatus). Compared to R. aculeatus, O. cubicus exhibited significantly lower detection thresholds for blue and achromatic grey, but not for green stimuli. Together, we find no support for five distinct spectral channels in the colour vision system in Australian boxfishes. Instead, these boxfish likely have trichromatic colour vision optimised for contrast detection, highlighting the importance of integrating multiple lines of investigation for accurately characterising animal colour perception.

Visual ecology, the study of how animals acquire and respond to visual information in nature, has grown rapidly over the past few decades. Research in this field has transformed our understanding of fundamental processes, such as the neurobiological basis of behavior and the diversification of species through sensory drive. The recent growth in the field has been accompanied by leaps in our understanding of the diversity of visual systems and in the development of novel technologies and techniques (for example, those allowing us to measure scenes and signals). With such growth, however, it is more important than ever to integrate wide perspectives and expertise to move the field forward in the most productive way. To that end, in summer 2024, 30 visual ecologists from around the world - spanning all career stages - met to discuss the state of the field. From that meeting, we identified two broad emerging themes in the study of visual ecology. (1) Can we further 'step inside' the perceptual experience of a non-human animal? (2) Can foundational 'rules' of vision and visual stimuli be identified? Although large questions such as these can feel unanswerable, this is where some of the most exciting discoveries in visual ecology remain to be made. Here, we outline eight relevant areas of research and identify ways in which researchers can bring us closer to answering these complex questions.

ABSTRACT 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, and 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 11 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.

Visually hunting predators drive the evolution and maintenance of bold visual defences, including aposematic signalling. Such visual signals must be encountered by predators in lighting conditions where the perception of colour patterns is possible. Therefore, aposematic colouration is predicted to evolve in species encountered by visually hunting predators during daytime, with diurnal species most at risk of visual predation. However, colour patterns in diurnal and nocturnal species are highly diverse, and little is known about the relationships between prey colouration and daytime activity. To investigate correlations between daytime activity and colour pattern phenotypes across species, we quantified colour patterns in 45 species ( n = 346 individuals) of eastern Australian sea slugs (Gastropoda: Heterobranchia). We used 158 descriptors of colour pattern morphology using the Quantitative Colour Pattern Analysis (QCPA) framework, which models pattern appearance using the visual system of a potential predator, a triggerfish ( Rhinecanthus aculeatus ). We then used phylogenetic factor analysis to identify a single factor that was strongly correlated with daytime activity, comprising 55 individual colour pattern descriptors. This dimension of pattern variation identified daytime activity in 87% of species in our dataset. We found that daytime activity in heterobranch sea slugs was associated with a distinct, highly descriptive set of spatiochromatic image statistics. Specifically, diurnal species were more boldly patterned than nocturnal species, exhibiting increased colour, luminance and pattern contrast, indicating the presence of visual signalling and, thus, visual predation as a key driver of heterobranch sea slug phenotype. Our study sheds light on the ecology and evolution of defensive colouration in sea slugs and provides a novel and robust workflow for comparative phylogenetic studies considering colour pattern spaces described by high‐dimensional datasets, which can be applied to other species and ecosystems.

Some species have evolved innovative solutions that allow them to thrive in extreme environments, including in habitats where temperature and ultraviolet radiation are high. Here we focus on the role of colour, particularly a blue carotenoprotein pigment, for survival in an environmentally stressful habitat. We show that blue Linckia laevigata seastars are common on tropical reef flats throughout the Indo-West Pacific, where at low tide, water temperatures can reach 40º C and water levels frequently drop < 0.5 m, sometimes resulting in animals being fully exposed to the air and ultraviolet radiation. Conversely, non-blue morphs live in water ≥ 1 m, where these stressors are ameliorated. Despite their different depth distribution, blue L. laevigata do not differ significantly from orange morphs in their ability to block ultraviolet radiation, which suggests that the blue carotenoprotein pigment is not a physical sunscreen. However, we show that saline extracts of skin, with intact blue carotenoproteins, demonstrate antioxidant capacity and in fact demonstrate more than twice as much oxygen radical absorbance capacity as acetone extracts where the carotenoids (mainly astaxanthin) and protein moiety have dissociated. Together with depth data, this suggests that the blue pigment provides protection from environmental stress through an increased capacity to delay, prevent or repair oxidative damage and is a key evolutionary innovation in L. laevigata.

Animal and plant colouration presents a striking dimension of phenotypic variation, the study of which has driven general advances in ecology, evolution, and animal behaviour. Quantitative Colour Pattern Analysis (QCPA) is a dynamic framework for analysing colour patterns through the eyes of non-human observers. However, its extensive array of user-defined image processing and analysis tools means image analysis is often time-consuming. This hinders the full use of analytical power provided by QCPA and its application to large datasets. Here, we offer a robust and comprehensive batch script, allowing users to automate many QCPA workflows. We also provide a complimentary set of useful R scripts for downstream data extraction and analysis. The presented batch processing extension will empower users to further utilise the analytical power of QCPA and facilitate the development of customised semi-automated workflows. Such quantitatively scaled workflows are crucial for exploring colour pattern spaces and developing ever-richer frameworks for analysing organismal colouration accounting for visual perception in animals other than humans. These advances will, in turn, facilitate testing hypotheses on the function and evolution of vision and signals at quantitative and qualitative scales, which are otherwise computationally unfeasible.

The selective factors that shape phenotypic diversity in prey communities with aposematic animals are diverse and coincide with similar diversity in the strength of underlying secondary defences. However, quantitative assessments of colour pattern variation and the strength of chemical defences in assemblages of aposematic species are lacking. We quantified colour pattern diversity using quantitative colour pattern analysis (QCPA) in 13 dorid nudibranch species (Infraorder: Doridoidei) that varied in the strength of their chemical defences. We accounted for the physiological properties of a potential predator’s visual system (a triggerfish, Rhinecanthus aculeatus ) and modelled the appearance of nudibranchs from multiple viewing distances (2 and 10 cm). We identified distinct colour pattern properties associated with the presence and strength of chemical defences. Specifically, increases in chemical defences indicated increases in colour pattern boldness (i.e. visual contrast elicited via either or potentially coinciding chromatic, achromatic and/or spatial contrast). Colour patterns were also less variable among species with chemical defences when compared to undefended species. Our results indicate correlations between secondary defences and diverse, bold colouration while showing that chemical defences coincide with decreased colour pattern variability among species. Our study suggests that complex spatiochromatic properties of colour patterns perceived by potential predators can be used to make inferences on the presence and strength of chemical defences.

In many animals, ultraviolet (UV) vision guides navigation, foraging, and communication, but few studies have addressed the contribution of UV signals to colour vision, or measured UV discrimination thresholds using behavioural experiments. Here, we tested UV colour vision in an anemonefish (Amphiprion ocellaris) using a five -channel (RGB-V-UV) LED display. We first determined that the maximal sensitivity of the A. ocellaris UV cone was -386 nm using microspectrophotometry. Three additional cone spectral sensitivities had maxima at -497, 515 and -535 nm. We then behaviourally measured colour discrimination thresholds by training anemonefish to distinguish a coloured target pixel from grey distractor pixels of varying intensity. Thresholds were calculated for nine sets of colours with and without UV signals. Using a tetrachromatic vision model, we found that anemonefish were better (i.e. discrimination thresholds were lower) at discriminating colours when target pixels had higher UV chromatic contrast. These colours caused a greater stimulation of the UV cone relative to other cone types. These findings imply that a UV component of colour signals and cues improves their detectability, which likely increases the prominence of anemonefish body patterns for communication and the silhouette of zooplankton prey.

The "escape and radiate" hypothesis predicts that once species have evolved aposematism, defended species can utilize more visually diverse visual backgrounds as they "escape" the need to be well camouflaged. This enables species to explore new ecological niches, resulting in increased diversification rates. To test this hypothesis "escape" component, we examined whether the background habitats of 12 nudibranch mollusk species differed among species depending on the presence and strength of chemical defenses. We obtained a rich array of color pattern statistics using quantitative color pattern analysis to analyze backgrounds viewed through the eyes of a potential predator (triggerfish, Rhinecanthus aculeatus). Color pattern analysis was done at viewing distances simulating an escalating predation sequence. We identified 4 latent factors comprising 17 noncorrelated color pattern parameters, which captured the among-species variability associated with differences in chemical defenses. We found that chemically defended species, indeed, were found on visually distinct backgrounds with increased color and luminance contrast, independent of viewing distance. However, we found no evidence for increased among-species background diversity coinciding with the presence and strength of chemical defenses. Our results agree with the "escape and radiate" hypothesis, suggesting that potent chemical defenses in Dorid nudibranchs coincide with spatiochromatic differences of visual background habitats perceived by potential predators.

Abstract Ultraviolet (UV) vision is widespread among teleost fishes, of which many exhibit UV skin colors for communication. However, aside from its role in mate selection, few studies have examined the information UV signaling conveys in other socio-behavioral contexts. Anemonefishes (subfamily, Amphiprioninae) live in a fascinating dominance hierarchy, in which a large female and male dominate over non-breeding subordinates, and body size is the primary cue for dominance. The iconic orange and white bars of anemonefishes are highly UV-reflective, and their color vision is well tuned to perceive the chromatic contrast of skin, which we show here decreases in the amount of UV reflectance with increasing social rank. To test the function of their UV-skin signals, we compared the outcomes of staged contests over dominance between size-matched Barrier Reef anemonefish (Amphiprion akindynos) in aquarium chambers viewed under different UV-absorbing filters. Fish under UV-blocking filters were more likely to win contests, where fish under no-filter or neutral-density filter were more likely to submit. For contests between fish in no-filter and neutral density filter treatments, light treatment had no effect on contest outcome (win/lose). We also show that sub-adults were more aggressive toward smaller juveniles placed under a UV filter than a neutral density filter. Taken together, our results show that UV reflectance or UV contrast in anemonefish can modulate aggression and encode dominant and submissive cues, when changes in overall intensity are controlled for.

Prey seldom rely on a single type of antipredator defence, often using multiple defences to avoid predation. In many cases, selection in different contexts may favour the evolution of multiple defences in a prey. However, a prey may use multiple defences to protect itself during a single predator encounter. Such "defence portfolios" that defend prey against a single instance of predation are distributed across and within successive stages of the predation sequence (encounter, detection, identification, approach (attack), subjugation and consumption). We contend that at present, our understanding of defence portfolio evolution is incomplete, and seen from the fragmentary perspective of specific sensory systems (e.g., visual) or specific types of defences (especially aposematism). In this review, we aim to build a comprehensive framework for conceptualizing the evolution of multiple prey defences, beginning with hypotheses for the evolution of multiple defences in general, and defence portfolios in particular. We then examine idealized models of resource trade-offs and functional interactions between traits, along with evidence supporting them. We find that defence portfolios are constrained by resource allocation to other aspects of life history, as well as functional incompatibilities between different defences. We also find that selection is likely to favour combinations of defences that have synergistic effects on predator behaviour and prey survival. Next, we examine specific aspects of prey ecology, genetics and development, and predator cognition that modify the predictions of current hypotheses or introduce competing hypotheses. We outline schema for gathering data on the distribution of prey defences across species and geography, determining how multiple defences are produced, and testing the proximate mechanisms by which multiple prey defences impact predator behaviour. Adopting these approaches will strengthen our understanding of multiple defensive strategies.

Aposematic signals visually advertise underlying anti-predatory defences in many species. They should be detectable (e.g. contrasting against the background) and bold (e.g. using internal pattern contrast) to enhance predator recognition, learning and memorization. However, the signalling function of aposematic colour patterns may be distance-dependent: signals may be undetectable from a distance to reduce increased attacks from naïve predators but bold when viewed up close. Using quantitative colour pattern analysis, we quantified the chromatic and achromatic detectability and boldness of colour patterns in 13 nudibranch species with variable strength of chemical defences in terms of unpalatability and toxicity, approximating the visual perception of a triggerfish (Rhinecanthus aculeatus) across a predation sequence (detection to subjugation). When viewed from an ecologically relevant distance of 30 cm, there were no differences in detectability and boldness between well-defended and undefended species. However, when viewed at closer distances (less than 30 cm), well-defended species were more detectable and bolder than undefended species. As distance increased, detectability decreased more significantly than boldness for defended species. For undefended species, boldness and detectability remained comparatively consistent, regardless of viewing distance. We provide evidence for distance-dependent signalling in aposematic nudibranchs and highlight the importance of distinguishing signal detectability from boldness in studies of aposematism.

ABSTRACT Animals use colour vision in a range of behaviours. Visual performance is limited by thresholds, which are set by noise in photoreceptors and subsequent neural processing. The receptor noise limited (RNL) model of colour discrimination is widely used for modelling colour vision and accounts well for experimental data from many species. In one of the most comprehensive tests yet of colour discrimination in a non-human species, we used Ishihara-style stimulus patterns to examine thresholds for 21 directions at five locations in colour space for the fish Rhinecanthus aculeatus. Thresholds matched RNL model predictions most closely for stimuli near the achromatic point, but exceeded predictions (indicating a decline in sensitivity) with distance from this point. Thresholds were also usually higher for saturation than for hue differences. These changes in colour threshold with colour space location and direction may give insight into photoreceptor non-linearities and post-receptoral mechanisms of colour vision in fish. Our results highlight the need for a cautious interpretation of the RNL model – especially for modelling colours that differ from one another in saturation (rather than hue), and for highly saturated colours distant from the achromatic point in colour space.

ABSTRACT Vision is used by animals to find food and mates, avoid predators, defend resources and navigate through complex habitats. Behavioural experiments are essential for understanding animals' perception but are often challenging and time-consuming; therefore, using species that can be trained easily for complex tasks is advantageous. Picasso triggerfish, Rhinecanthus aculeatus, have been used in many behavioural studies investigating vision and navigation. However, little is known about the molecular and anatomical basis of their visual system. We addressed this knowledge gap here and behaviourally tested achromatic and chromatic acuity. In terms of visual opsins, R. aculeatus possessed one rod opsin gene (RH1) and at least nine cone opsins: one violet-sensitive SWS2B gene, seven duplicates of the blue–green-sensitive RH2 gene (RH2A, RH2B, RH2C1-5) and one red-sensitive LWS gene. However, only five cone opsins were expressed: SWS2B expression was consistent, while RH2A, RH2C-1 and RH2C-2 expression varied depending on whether fish were sampled from the field or aquaria. Levels of LWS expression were very low. Using fluorescence in situ hybridisation, we found SWS2B was expressed exclusively in single cones, whereas RH2A and RH2Cs were expressed in opposite double cone members. Anatomical resolution estimated from ganglion cell densities was 6.8 cycles per degree (cpd), which was significantly higher than values obtained from behavioural testing for black-and-white achromatic stimuli (3.9 cpd) and chromatic stimuli (1.7–1.8 cpd). These measures were twice as high as previously reported. This detailed information on their visual system will help inform future studies with this emerging focal species.

In this paper, we investigate the presence of latrunculin A in the outer rim of a nudibranch Chromodoris kuiteri and show that by combining ultrathin cryosection methods with MALDI MSI we can achieve improved lateral (x and y) resolution and very high resolution in the z dimension by virtue of the ultrathin 200 nm thin cryosections. We also demonstrate that a post ionization laser increases sensitivity. Recent advances in MALDI source design have improved the lateral resolution (x and y) and sensitivity during MSI. Taken together, very high z resolution, from ultrathin sections, and improved lateral (x and y) resolution will allow for subcellular molecular imaging with the potential for subcellular 3D volume reconstruction.

Defensive chemicals are used by plants and animals to reduce the risk of predation through different mechanisms, including toxins that cause injury and harm (weapons) and unpalatable or odiferous compounds that prevent attacks (deterrents). However, whether effective defences are both toxins and deterrents, or work in just one modality is often unclear. In this study, our primary aim was to determine whether defensive compounds stored by nudibranch molluscs acted as weapons (in terms of being toxic), deterrents (in terms of being distasteful) or both. Our secondary aim was to investigate the response of different taxa to these defensive compounds. To do this, we identified secondary metabolites in 30 species of nudibranch molluscs and investigated their deterrent properties using antifeedant assays with three taxa: rock pool shrimp, Palaemon serenus, and two fish species: triggerfish Rhinecanthus aculeatus and toadfish Tetractenos hamiltoni. We compared these results to toxicity assays using brine shrimp Artemia sp. and previously published toxicity data with a damselfish Chromis viridis. Overall, we found no clear relationship between palatability and toxicity, but instead classified defensive compounds into the following categories: Class I & II-highly unpalatable and highly toxic; Class I-weakly unpalatable and highly toxic; Class II-highly unpalatable but weakly toxic; WR (weak response)-weakly unpalatable and weakly toxic. We also found eight extracts from six species that did not display activity in any assays indicating they may have very limited chemical defensive mechanisms (NR, no response). We found that the different classes of secondary metabolites were similarly unpalatable to fish and shrimp, except extracts from Phyllidiidae nudibranchs (isonitriles) that were highly unpalatable to shrimp but weakly unpalatable to fish. Our results pave the way towards better understanding how animal chemical defences work against a variety of predators. We highlight the need to disentangle weapons and deterrents in future work on anti-predator defences to better understand the foraging decisions faced by predators, the resultant selection pressures imposed on prey and the evolution of different anti-predator strategies.

Edge detection is important for object detection and recognition. However, we do not know whether edge statistics accurately predict the detection of prey by potential predators. This is crucial given the growing availability of image analysis software and their application across non-human visual systems. Here, we investigated whether Boundary Strength Analysis (BSA), Local Edge Intensity Analysis (LEIA) and the Gabor edge disruption ratio (GabRat) could predict the speed and success with which triggerfish (Rhinecanthus aculeatus) detected patterned circular stimuli against a noisy visual background, in both chromatic and achromatic presentations. We found various statistically significant correlations between edge statistics and detection speed depending on treatment and viewing distance; however, individual pattern statistics only explained up to 2% of the variation in detection time, and up to 6% when considering edge statistics simultaneously. We also found changes in fish response over time. While highlighting the importance of spatial acuity and relevant viewing distances in the study of visual signals, our results demonstrate the importance of considering explained variation when interpreting colour pattern statistics in behavioural experiments. We emphasize the need for statistical approaches suitable for investigating task-specific predictive relationships and ecological effects when considering animal behaviour. This is particularly important given the ever-increasing dimensionality and size of datasets in the field of visual ecology.

Edge detection is important for object detection and recognition. However, we do not know whether edge statistics predict the detection of prey by non-human predators. Understanding the link between image statistics and animal behaviour is crucial and of increasing importance given the growing availability of image analyses and their application across non-human visual systems. Here, we investigated whether Boundary Strength Analysis (BSA), Local Edge Intensity Analysis (LEIA) and the Gabor Ratio (GabRat) could predict the speed and success with which triggerfish ( Rhinecanthus aculeatus ) detected patterned circular stimuli against a noisy visual background, in both chromatic and achromatic presentations. We found that individual pattern statistics could only explain up to 2% of the variation in detection time, whereas PCA regression analysis considering all edge statistics simultaneously explained up to 6% of the variation. This suggests that other factors explained more behavioural variation than individual edge statistics. Furthermore, different statistics significantly correlated with detection speed depending on treatment, viewing distance, and changes in fish response over time, while highlighting the importance of considering spatial acuity and relevant viewing distances in the study of visual signals. Our results demonstrate the need for broad and unbiased approaches for identifying task-specific predictive relationships between pattern statistics and animal behaviour using image statistics capturing different aspects of colour patterns. We require robust statistical approaches to investigate correlations between ecological effect and the ever-increasing dimensionality and size of datasets in the field of visual ecology, rather than pre-emptively narrowing down the choice of image statistics unless warranted by specific hypotheses. Summary statement Correlations between edge detecting colour pattern statistics and animal behaviour are complex. Specifically, correlations are unlikely to be explained by single image statistics and depend upon observer distance. ### Competing Interest Statement The authors have declared no competing interest.