Pupil dilation and constriction in the skate Leucoraja erinacea in a simulated natural light field.

The skate Leucoraja erinacea has an elaborately shaped pupil, whose characteristics and functions have received little attention. The goal of our study was to investigate the pupil response in relation to natural ambient light intensities. First, we took a recently developed sensory-ecological approach, which gave us a tool for creating a controlled light environment for behavioural work: during a field survey, we collected a series of calibrated natural habitat images from the perspective of the skates' eyes. From these images, we derived a vertical illumination profile using custom-written software for quantification of the environmental light field (ELF). After collecting and analysing these natural light field data, we created an illumination set-up in the laboratory, which closely simulated the natural vertical light gradient that skates experience in the wild and tested the light responsiveness - in particular the extent of dilation - of the skate pupil to controlled changes in this simulated light field. Additionally, we measured pupillary dilation and constriction speeds. Our results confirm that the skate pupil changes from nearly circular under low light to a series of small triangular apertures under bright light. A linear regression analysis showed a trend towards smaller skates having a smaller dynamic range of pupil area (dilation versus constriction ratio around 4-fold), and larger skates showing larger ranges (around 10- to 20-fold). Dilation took longer than constriction (between 30 and 45 min for dilation; less than 20 min for constriction), and there was considerable individual variation in dilation/constriction time. We discuss our findings in terms of the visual ecology of L. erinacea and consider the importance of accurately simulating natural light fields in the laboratory.

Elaborate pupils in skates may help camouflage the eye.

The skate Leucoraja erinacea is a bottom-dweller that buries into the substrate with its eyes protruding, revealing elaborately shaped pupils. It has been suggested that such pupil shapes may camouflage the eye, yet this has never been tested. Here, we asked whether skate pupils dilate or constrict depending on background spatial frequency. In experiment 1, the skates' pupillary response to three artificial checkerboards of different spatial frequencies was recorded. Results showed that pupils did not change in response to spatial frequency. In experiment 2, in which skates buried into three natural substrates of different spatial frequencies, such that their eyes protruded, pupils showed a subtle but statistically significant response to changes in substrate spatial frequency. Although light intensity is the primary factor determining pupil dilation, our results show that pupils also change depending on the spatial frequency of natural substrates, which suggests that pupils may aid in camouflaging the eye.

Quantification of cuttlefish (Sepia officinalis) camouflage: a study of color and luminance using in situ spectrometry.

Cephalopods are renowned for their ability to adaptively camouflage on diverse backgrounds. Sepia officinalis camouflage body patterns have been characterized spectrally in the laboratory but not in the field due to the challenges of dynamic natural light fields and the difficulty of using spectrophotometric instruments underwater. To assess cuttlefish color match in their natural habitats, we studied the spectral properties of S. officinalis and their backgrounds on the Aegean coast of Turkey using point-by-point in situ spectrometry. Fifteen spectrometry datasets were collected from seven cuttlefish; radiance spectra from animal body components and surrounding substrates were measured at depths shallower than 5 m. We quantified luminance and color contrast of cuttlefish components and background substrates in the eyes of hypothetical di- and trichromatic fish predators. Additionally, we converted radiance spectra to sRGB color space to simulate their in situ appearance to a human observer. Within the range of natural colors at our study site, cuttlefish closely matched the substrate spectra in a variety of body patterns. Theoretical calculations showed that this effect might be more pronounced at greater depths. We also showed that a non-biological method ("Spectral Angle Mapper"), commonly used for spectral shape similarity assessment in the field of remote sensing, shows moderate correlation to biological measures of color contrast. This performance is comparable to that of a traditional measure of spectral shape similarity, hue and chroma. This study is among the first to quantify color matching of camouflaged cuttlefish in the wild.

Cuttlefish use visual cues to determine arm postures for camouflage.

To achieve effective visual camouflage, prey organisms must combine cryptic coloration with the appropriate posture and behaviour to render them difficult to be detected or recognized. Body patterning has been studied in various taxa, yet body postures and their implementation on different backgrounds have seldom been studied experimentally. Here, we provide the first experimental evidence that cuttlefish (Sepia officinalis), masters of rapid adaptive camouflage, use visual cues from adjacent visual stimuli to control arm postures. Cuttlefish were presented with a square wave stimulus (period = 0.47 cm; black and white stripes) that was angled 0°, 45° or 90° relative to the animals' horizontal body axis. Cuttlefish positioned their arms parallel, obliquely or transversely to their body axis according to the orientation of the stripes. These experimental results corroborate our field observations of cuttlefish camouflage behaviour in which flexible, precise arm posture is often tailored to match nearby objects. By relating the cuttlefishes' visual perception of backgrounds to their versatile postural behaviour, our results highlight yet another of the many flexible and adaptive anti-predator tactics adopted by cephalopods.

How does the blue-ringed octopus (Hapalochlaena lunulata) flash its blue rings?

The blue-ringed octopus (Hapalochlaena lunulata), one of the world's most venomous animals, has long captivated and endangered a large audience: children playing at the beach, divers turning over rocks, and biologists researching neurotoxins. These small animals spend much of their time in hiding, showing effective camouflage patterns. When disturbed, the octopus will flash around 60 iridescent blue rings and, when strongly harassed, bite and deliver a neurotoxin that can kill a human. Here, we describe the flashing mechanism and optical properties of these rings. The rings contain physiologically inert multilayer reflectors, arranged to reflect blue-green light in a broad viewing direction. Dark pigmented chromatophores are found beneath and around each ring to enhance contrast. No chromatophores are above the ring; this is unusual for cephalopods, which typically use chromatophores to cover or spectrally modify iridescence. The fast flashes are achieved using muscles under direct neural control. The ring is hidden by contraction of muscles above the iridophores; relaxation of these muscles and contraction of muscles outside the ring expose the iridescence. This mechanism of producing iridescent signals has not previously been reported in cephalopods and we suggest that it is an exceptionally effective way to create a fast and conspicuous warning display.

Photoreceptors in skate are arranged to allow for a broad horizontal field of view.

Studying retinal specializations offers insights into eye functionality and visual ecology. Using light microscopic techniques, including retinal whole-mounts, we investigated photoreceptor densities in the retina of the skate Leucoraja erinacea. We show that photoreceptors are not sized or oriented in the same way, and that they are not evenly distributed across the retina. There was a dorsally located horizontal visual streak with increased photoreceptor density, with additional local maxima in which densities were highest. Photoreceptors were longest and thinnest inside this visual streak, becoming shorter and thicker toward the periphery and toward the ventral retina. Furthermore, in the peripheral retinal parts, photoreceptors (particularly the outer segments) were noticeably tilted with respect to the retinal long axis. In order to understand how photoreceptors are tilted inside the eye, we used computerized tomography (CT) and micro-CT, to obtain geometrical dimensions of the whole skate eye. These CT/micro-CT data provided us with the outlines of the skate eye and the location of the retina and this enabled us to reconstruct how photoreceptors tilt in an intact eye. Findings were analyzed relative to previously published ganglion cell distributions in this species, showing a posteriorly located retinal area with photoreceptor: ganglion cell convergence as low as 39:1. Some peripheral areas showed ratios as high as 391:1. We frame our findings in terms of the animal's anatomy: body and eye shape, specifically the location of the tapetum, as well as the visual demands associated with lifestyle and habitat type. A speculative function in polarization sensitivity is discussed.

Cuttlefish dynamic camouflage: responses to substrate choice and integration of multiple visual cues.

Prey camouflage is an evolutionary response to predation pressure. Cephalopods have extensive camouflage capabilities and studying them can offer insight into effective camouflage design. Here, we examine whether cuttlefish, Sepia officinalis, show substrate or camouflage pattern preferences. In the first two experiments, cuttlefish were presented with a choice between different artificial substrates or between different natural substrates. First, the ability of cuttlefish to show substrate preference on artificial and natural substrates was established. Next, cuttlefish were offered substrates known to evoke three main camouflage body pattern types these animals show: Uniform or Mottle (function by background matching); or Disruptive. In a third experiment, cuttlefish were presented with conflicting visual cues on their left and right sides to assess their camouflage response. Given a choice between substrates they might encounter in nature, we found no strong substrate preference except when cuttlefish could bury themselves. Additionally, cuttlefish responded to conflicting visual cues with mixed body patterns in both the substrate preference and split substrate experiments. These results suggest that differences in energy costs for different camouflage body patterns may be minor and that pattern mixing and symmetry may play important roles in camouflage.

Night vision by cuttlefish enables changeable camouflage.

Because visual predation occurs day and night, many predators must have good night vision. Prey therefore exhibit antipredator behaviours in very dim light. In the field, the giant Australian cuttlefish (Sepia apama) assumes camouflaged body patterns at night, each tailored to its immediate environment. However, the question of whether cuttlefish have the perceptual capability to change their camouflage at night (as they do in day) has not been addressed. In this study, we: (1) monitored the camouflage patterns of Sepia officinalis during the transition from daytime to night-time using a natural daylight cycle and (2) tested whether cuttlefish on a particular artificial substrate change their camouflage body patterns when the substrate is changed under dim light (down to starlight, 0.003 lux) in a controlled light field in a dark room setting. We found that cuttlefish camouflage patterns are indeed adaptable at night: animals responded to a change in their visual environment with the appropriate body pattern change. Whether to deceive their prey or predators, cuttlefish use their excellent night vision to perform adaptive camouflage in dim light.

Mottle camouflage patterns in cuttlefish: quantitative characterization and visual background stimuli that evoke them.

Cuttlefish and other cephalopods achieve dynamic background matching with two general classes of body patterns: uniform (or uniformly stippled) patterns and mottle patterns. Both pattern types have been described chiefly by the size scale and contrast of their skin components. Mottle body patterns in cephalopods have been characterized previously as small-to-moderate-scale light and dark skin patches (i.e. mottles) distributed somewhat evenly across the body surface. Here we move beyond this commonly accepted qualitative description by quantitatively measuring the scale and contrast of mottled skin components and relating these statistics to specific visual background stimuli (psychophysics approach) that evoke this type of background-matching pattern. Cuttlefish were tested on artificial and natural substrates to experimentally determine some primary visual background cues that evoke mottle patterns. Randomly distributed small-scale light and dark objects (or with some repetition of small-scale shapes/sizes) on a lighter substrate with moderate contrast are essential visual cues to elicit mottle camouflage patterns in cuttlefish. Lowering the mean luminance of the substrate without changing its spatial properties can modulate the mottle pattern toward disruptive patterns, which are of larger scale, different shape and higher contrast. Backgrounds throughout nature consist of a continuous range of spatial scales; backgrounds with medium-sized light/dark patches of moderate contrast are those in which cuttlefish Mottle patterns appear to be the most frequently observed.

Evidence for distributed light sensing in the skin of cuttlefish, Sepia officinalis.

We report that the skin of cuttlefish, Sepia officinalis, contains opsin transcripts suggesting a possible role of distributed light sensing for dynamic camouflage and signalling. The mRNA coding for opsin from various body regions was amplified and sequenced, and gene expression was detected in fin and ventral skin samples. The amino acid sequence of the opsin polypeptide that these transcripts would produce was identical in retina and fin tissue samples, but the ventral skin opsin transcripts differed by a single amino acid. The diverse camouflage and signalling body patterns of cephalopods are visually controlled, and these findings suggest a possible additional mechanism of light sensing and subsequent skin patterning. Cuttlefish, along with a number of other cephalopod species, have been shown to be colour-blind. Since the opsin in the fin is identical to that of the retina (λmax=492 nm), and the ventral transcripts are also unlikely to be spectrally different, colour discrimination by the skin opsins is unlikely. However, spectral discrimination could be provided by involving other skin structures (chromatophores and iridophores), which produce changeable colours and patterns. This 'distributed sensing' could supplement the otherwise visually driven dynamic camouflage system by assisting with colour or brightness matching to adjacent substrates.

Cuttlefish use visual cues to control three-dimensional skin papillae for camouflage.

Cephalopods (octopus, squid and cuttlefish) are known for their camouflage. Cuttlefish Sepia officinalis use chromatophores and light reflectors for color change, and papillae to change three-dimensional physical skin texture. Papillae vary in size, shape and coloration; nine distinct sets of papillae are described here. The objective was to determine whether cuttlefish use visual or tactile cues to control papillae expression. Cuttlefish were placed on natural substrates to evoke the three major camouflage body patterns: Uniform/Stipple, Mottle and Disruptive. Three versions of each substrate were presented: the actual substrate, the actual substrate covered with glass (removes tactile information) and a laminated photograph of the substrate (removes tactile and three-dimensional information because depth-of-field information is unavailable). No differences in Small dorsal papillae or Major lateral mantle papillae expression were observed among the three versions of each substrate. Thus, visual (not tactile) cues drive the expression of papillae in S. officinalis. Two sets of papillae (Major lateral mantle papillae and Major lateral eye papillae) showed irregular responses; their control requires future investigation. Finally, more Small dorsal papillae were shown in Uniform/Stipple and Mottle patterns than in Disruptive patterns, which may provide clues regarding the visual mechanisms of background matching versus disruptive coloration.

Mechanisms and behavioural functions of structural coloration in cephalopods.

Octopus, squid and cuttlefish are renowned for rapid adaptive coloration that is used for a wide range of communication and camouflage. Structural coloration plays a key role in augmenting the skin patterning that is produced largely by neurally controlled pigmented chromatophore organs. While most iridescence and white scattering is produced by passive reflectance or diffusion, some iridophores in squid are actively controlled via a unique cholinergic, non-synaptic neural system. We review the recent anatomical and experimental evidence regarding the mechanisms of reflection and diffusion of light by the different cell types (iridophores and leucophores) of various cephalopod species. The structures that are responsible for the optical effects of some iridophores and leucophores have recently been shown to be proteins. Optical interactions with the overlying pigmented chromatophores are complex, and the recent measurements are presented and synthesized. Polarized light reflected from iridophores can be passed through the chromatophores, thus enabling the use of a discrete communication channel, because cephalopods are especially sensitive to polarized light. We illustrate how structural coloration contributes to the overall appearance of the cephalopods during intra- and interspecific behavioural interactions including camouflage.

Cuttlefish camouflage: the effects of substrate contrast and size in evoking uniform, mottle or disruptive body patterns.

Cuttlefish are cephalopod molluscs that achieve dynamic camouflage by rapidly extracting visual information from the background and neurally implementing an appropriate skin (or body) pattern. We investigated how cuttlefish body patterning responses are influenced by contrast and spatial scale by varying the contrast and the size of checkerboard backgrounds. We found that: (1) at high contrast levels, cuttlefish body patterning depended on check size; (2) for low contrast levels, body patterning was independent of "check" size; and (3) on the same check size, cuttlefish fine-tuned the contrast and fine structure of their body patterns, in response to small contrast changes in the background. Furthermore, we developed an objective, automated method of assessing cuttlefish camouflage patterns that quantitatively differentiated the three body patterns of uniform/stipple, mottle and disruptive. This study draws attention to the key roles played by background contrast and particle size in determining an effective camouflage pattern.

Retinal specializations and visual ecology in an animal with an extremely elaborate pupil shape: the little skate Leucoraja (Raja) erinacea Mitchell, 1825.

Investigating retinal specializations offers insights into eye functionality. Using retinal wholemount techniques, we investigated the distribution of retinal ganglion cells in the Little skate Leucoraja erinacea by (a) dye-backfilling into the optic nerve prior to retinal wholemounting; (b) Nissl-staining of retinal wholemounts. Retinas were examined for regional specializations (higher numbers) of ganglion cells that would indicate higher visual acuity in those areas. Total ganglion cell number were low compared to other elasmobranchs (backfilled: average 49,713 total ganglion cells, average peak cell density 1,315 ganglion cells mm-2 ; Nissl-stained: average 47,791 total ganglion cells, average peak cell density 1,319 ganglion cells mm-2 ). Ganglion cells fit into three size categories: small (5-20 µm); medium (20-30 µm); large: (≥ 30 µm), and they were not homogeneously distributed across the retina. There was a dorsally located horizontal visual streak with increased ganglion cell density; additionally, there were approximately three local maxima in ganglion cell distribution (potential areae centrales) within this streak in which densities were highest. Using computerized tomography (CT) and micro-CT, geometrical dimensions of the eye were obtained. Combined with ganglion cell distributions, spatial resolving power was determined to be between 1.21 and 1.37 cycles per degree. Additionally, photoreceptor sizes across different retinal areas varied; photoreceptors were longest within the horizontal visual streak. Variations in the locations of retinal specializations appear to be related to the animal's anatomy: shape of the head and eyes, position of eyes, location of tapetum, and shape of pupil, as well as the visual demands associated with lifestyle and habitat type.

White reflection from cuttlefish skin leucophores.

The highly diverse and changeable body patterns of cephalopods require the production of whiteness of varying degrees of brightness for their large repertoire of communication and camouflage behaviors. Leucophores are structural reflectors that produce whiteness in cephalopods; they are dermal aggregates of numerous leucocytes containing spherical leucosomes ranging in diameter from 200-2000 nm. In Sepia officinalis leucophores, leucocytes always occur in various combinations with iridocytes, cells containing plates that function as Bragg stacks to reflect light of particular wavelengths. Both spheres and plates contain the high-refractive-index protein reflectin. Four leucophore skin-patterning components were investigated morphologically and with spectrometry. In descending order of brightness they are: white fin spots, White zebra bands, White square, and White head bar. Different densities, thicknesses and proportions of leucocytes and iridocytes were correlated with the relative brightness measurements of the skin. That is, White fin spots and White zebra bands had leucocytes of the highest density, the greatest number of reflective cell layers, and the highest proportion of leucocytes to iridocytes. In contrast, the White square and White head bar had the lowest density of reflective cells, fewer cell layers and the lowest ratios of leucocytes to iridocytes. Leucophores are white in white light, yet reflect whatever colors are in the available light field: e.g. red in red light, green in green light, etc. Leucophores are physiologically passive, thus their ultrastructure alone is capable of diffusing all ambient wavelengths in all directions, regardless of the angle of incident light. However, the specific optical contributions of spherical leucosomes versus the associated plate-like iridosomes in producing whiteness versus brightness are yet to be determined. This study reveals complex morphological arrangements that produce white structural coloration for different brightnesses of skin by differentially combining spheres and plates.

Changeable camouflage: how well can flounder resemble the colour and spatial scale of substrates in their natural habitats?

Flounder change colour and pattern for camouflage. We used a spectrometer to measure reflectance spectra and a digital camera to capture body patterns of two flounder species camouflaged on four natural backgrounds of different spatial scale (sand, small gravel, large gravel and rocks). We quantified the degree of spectral match between flounder and background relative to the situation of perfect camouflage in which flounder and background were assumed to have identical spectral distribution. Computations were carried out for three biologically relevant observers: monochromatic squid, dichromatic crab and trichromatic guitarfish. Our computations present a new approach to analysing datasets with multiple spectra that have large variance. Furthermore, to investigate the spatial match between flounder and background, images of flounder patterns were analysed using a custom program originally developed to study cuttlefish camouflage. Our results show that all flounder and background spectra fall within the same colour gamut and that, in terms of different observer visual systems, flounder matched most substrates in luminance and colour contrast. Flounder matched the spatial scales of all substrates except for rocks. We discuss findings in terms of flounder biology; furthermore, we discuss our methodology in light of hyperspectral technologies that combine high-resolution spectral and spatial imaging.

Color blindness and contrast perception in cuttlefish (Sepia officinalis) determined by a visual sensorimotor assay.

We tested color perception based upon a robust behavioral response in which cuttlefish (Sepia officinalis) respond to visual stimuli (a black and white checkerboard) with a quantifiable, neurally controlled motor response (a body pattern). In the first experiment, we created 16 checkerboard substrates in which 16 grey shades (from white to black) were paired with one green shade (matched to the maximum absorption wavelength of S. officinalis' sole visual pigment, 492 nm), assuming that one of the grey shades would give a similar achromatic signal to the tested green. In the second experiment, we created a checkerboard using one blue and one yellow shade whose intensities were matched to the cuttlefish's visual system. In both assays it was tested whether cuttlefish would show disruptive coloration on these checkerboards, indicating their ability to distinguish checkers based solely on wavelength (i.e., color). Here, we show clearly that cuttlefish must be color blind, as they showed non-disruptive coloration on the checkerboards whose color intensities were matched to the Sepia visual system, suggesting that the substrates appeared to their eyes as uniform backgrounds. Furthermore, we show that cuttlefish are able to perceive objects in their background that differ in contrast by approximately 15%. This study adds support to previous reports that S. officinalis is color blind, yet the question of how cuttlefish achieve "color-blind camouflage" in chromatically rich environments still remains.

Tactical decisions for changeable cuttlefish camouflage: visual cues for choosing masquerade are relevant from a greater distance than visual cues used for background matching.

Cuttlefish use multiple camouflage tactics to evade their predators. Two common tactics are background matching (resembling the background to hinder detection) and masquerade (resembling an uninteresting or inanimate object to impede detection or recognition). We investigated how the distance and orientation of visual stimuli affected the choice of these two camouflage tactics. In the current experiments, cuttlefish were presented with three visual cues: 2D horizontal floor, 2D vertical wall, and 3D object. Each was placed at several distances: directly beneath (in a circle whose diameter was one body length (BL); at zero BL [(0BL); i.e., directly beside, but not beneath the cuttlefish]; at 1BL; and at 2BL. Cuttlefish continued to respond to 3D visual cues from a greater distance than to a horizontal or vertical stimulus. It appears that background matching is chosen when visual cues are relevant only in the immediate benthic surroundings. However, for masquerade, objects located multiple body lengths away remained relevant for choice of camouflage.

The structure-function relationships of a natural nanoscale photonic device in cuttlefish chromatophores.

Cuttlefish, Sepia officinalis, possess neurally controlled, pigmented chromatophore organs that allow rapid changes in skin patterning and coloration in response to visual cues. This process of adaptive coloration is enabled by the 500% change in chromatophore surface area during actuation. We report two adaptations that help to explain how colour intensity is maintained in a fully expanded chromatophore when the pigment granules are distributed maximally: (i) pigment layers as thin as three granules that maintain optical effectiveness and (ii) the presence of high-refractive-index proteins-reflectin and crystallin-in granules. The latter discovery, combined with our finding that isolated chromatophore pigment granules fluoresce between 650 and 720 nm, refutes the prevailing hypothesis that cephalopod chromatophores are exclusively pigmentary organs composed solely of ommochromes. Perturbations to granular architecture alter optical properties, illustrating a role for nanostructure in the agile, optical responses of chromatophores. Our results suggest that cephalopod chromatophore pigment granules are more complex than homogeneous clusters of chromogenic pigments. They are luminescent protein nanostructures that facilitate the rapid and sophisticated changes exhibited in dermal pigmentation.

The W-shaped pupil in cuttlefish (Sepia officinalis): functions for improving horizontal vision.

The eyes of cuttlefish (Sepia officinalis) have a modified horizontal slit-pupil with a distinctive W-shape in bright light, while in darkness the pupil is circular. Two suggestions have previously been made for a function of the W-shape: (1) camouflaging the eye; (2) providing distance information. Since neither of these suggestions can fully explain the function of this pupil across the entire visual field, particularly the frontal and caudal periphery, we re-addressed the question of its functional significance. We took infra-red images of the eyes of live S. officinalis at different light intensities and from different viewing angles. This allowed us to determine the shape and light-admitting area of the pupil for different parts of the visual field. Our data show that the W-shaped pupil projects a blurred "W" directly onto the retina and that it effectively operates as vertical slits for the frontal and caudal parts of the visual field. We also took images of the natural habitat of S. officinalis and calculated the average vertical brightness distribution in the visual habitat. Computing a retinal illumination map shows that the W-shaped pupil is effective in balancing a vertically uneven light field: The constricted pupil reduces light from the dorsal part of the visual field significantly more than it reduces light from the horizontal band. This will cut the amount of direct sunlight that is scattered by the lens and ocular media, and thus improve image contrast particularly for the dimmer parts of the scene. We also conclude that the pupil provides even attenuation along the horizontal band, whereas a circular pupil would attenuate the image relatively more in the important frontal and caudal periphery of the visual field.