Networks of linked radial muscles could influence dynamic skin patterning of squid chromatophores.

The visibility of cephalopod chromatophore organs is regulated dynamically by rosettes of obliquely striated radial muscles that dilate or relax the diameter of a central pigmented sacculus in 100-300 ms. Each of the several dozen muscles has a flared proximal end that adheres tightly to its pigmented sacculus and an extremely elongated distal end which branches into single fibrils that anchor into the dermis. This geometry provides ample opportunity for overlap of the many muscles from neighboring chromatophores. The temporal activity of these muscles has been believed to be patterned exclusively by monosynaptic projections from sets of efferent motor axons originating in the chromatophore lobes of the suboesophageal brain. Based on historical observations that distal radial muscles from some chromatophores appear to extend closely to muscles from other chromatophores, we asked whether radial muscles actually make specialized contacts. Using 3D electron microscopy of Doryteuthis pealeii mantle skin, we discovered tight putatively functional muscle-to-muscle contacts between radial muscles from different chromatophores, including elaborate sets of axonal processes located adjacent to those myo-myo junctions. These detailed ultrastructural findings demonstrate auxiliary anatomical routes for radial muscle activation and suggest plausible mechanisms whereby local physical synchronization and axo-axonic processing in the periphery can contribute to chromatophore pattern dynamics such as "passing cloud."

Dynamic pigmentary and structural coloration within cephalopod chromatophore organs.

Chromatophore organs in cephalopod skin are known to produce ultra-fast changes in appearance for camouflage and communication. Light-scattering pigment granules within chromatocytes have been presumed to be the sole source of coloration in these complex organs. We report the discovery of structural coloration emanating in precise register with expanded pigmented chromatocytes. Concurrently, using an annotated squid chromatophore proteome together with microscopy, we identify a likely biochemical component of this reflective coloration as reflectin proteins distributed in sheath cells that envelop each chromatocyte. Additionally, within the chromatocytes, where the pigment resides in nanostructured granules, we find the lens protein Ω- crystallin interfacing tightly with pigment molecules. These findings offer fresh perspectives on the intricate biophotonic interplay between pigmentary and structural coloration elements tightly co-located within the same dynamic flexible organ - a feature that may help inspire the development of new classes of engineered materials that change color and 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.

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.