Neural control of cephalopod camouflage.

In Die Another Day, James Bond receives an Aston Martin that can render itself invisible by dynamically reproducing the surroundings on the car's "polymer skin". In what is widely regarded as the worst Bond movie ever, the invisible car scene is cited as the moment the plot plunges into the truly absurd. But what if nature had actually invented such a technology, and did so hundreds of millions of years ago? The coleoid cephalopods - octopus, cuttlefish and squid - are living examples of dynamic camouflage. Their skin is covered with a high-resolution array of 'cellular pixels' (chromatophores) that are controlled by the brain. To disappear into their surroundings, cephalopods recreate an approximation of their environment on their skin by activating different combinations of colored chromatophores. However, unlike the fictional Bond car, whose surface is coated in tiny cameras to detect the environment, cephalopods don't see the world with their skin. Instead, the visual world is detected by the eyes, processed in the brain, and then used to activate motor commands that direct the skin's camouflage pattern. Thus, cephalopod skin patterns are an external manifestation of their internal perception of the world. How do cephalopods approximate the world with their skin? What can this teach us about how brains work? And which neurobiological tools will be needed to uncover the neural basis of camouflage?

A brain atlas for the camouflaging dwarf cuttlefish, Sepia bandensis.

The coleoid cephalopods (cuttlefish, octopus, and squid) are a group of soft-bodied marine mollusks that exhibit an array of interesting biological phenomena, including dynamic camouflage, complex social behaviors, prehensile regenerating arms, and large brains capable of learning, memory, and problem-solving.1,2,3,4,5,6,7,8,9,10 The dwarf cuttlefish, Sepia bandensis, is a promising model cephalopod species due to its small size, substantial egg production, short generation time, and dynamic social and camouflage behaviors.11 Cuttlefish dynamically camouflage to their surroundings by changing the color, pattern, and texture of their skin. Camouflage is optically driven and is achieved by expanding and contracting hundreds of thousands of pigment-filled saccules (chromatophores) in the skin, which are controlled by motor neurons emanating from the brain. We generated a dwarf cuttlefish brain atlas using magnetic resonance imaging (MRI), deep learning, and histology, and we built an interactive web tool (https://www.cuttlebase.org/) to host the data. Guided by observations in other cephalopods,12,13,14,15,16,17,18,19,20 we identified 32 brain lobes, including two large optic lobes (75% the total volume of the brain), chromatophore lobes whose motor neurons directly innervate the chromatophores of the color-changing skin, and a vertical lobe that has been implicated in learning and memory. The brain largely conforms to the anatomy observed in other Sepia species and provides a valuable tool for exploring the neural basis of behavior in the experimentally facile dwarf cuttlefish.

Molecular characterization of cell types in the squid Loligo vulgaris.

Cephalopods are set apart from other mollusks by their advanced behavioral abilities and the complexity of their nervous systems. Because of the great evolutionary distance that separates vertebrates from cephalopods, it is evident that higher cognitive features have evolved separately in these clades despite the similarities that they share. Alongside their complex behavioral abilities, cephalopods have evolved specialized cells and tissues, such as the chromatophores for camouflage or suckers to grasp prey. Despite significant progress in genome and transcriptome sequencing, the molecular identities of cell types in cephalopods remain largely unknown. We here combine single-cell transcriptomics with in situ gene expression analysis to uncover cell type diversity in the European squid Loligo vulgaris. We describe cell types that are conserved with other phyla such as neurons, muscles, or connective tissues but also cephalopod-specific cells, such as chromatophores or sucker cells. Moreover, we investigate major components of the squid nervous system including progenitor and developing cells, differentiated cells of the brain and optic lobes, as well as sensory systems of the head. Our study provides a molecular assessment for conserved and novel cell types in cephalopods and a framework for mapping the nervous system of L. vulgaris.

The physiological cost of colour change: evidence, implications and mitigations.

Animals benefit from phenotypic plasticity in changing environments, but this can come at a cost. Colour change, used for camouflage, communication, thermoregulation and UV protection, represents one of the most common plastic traits in nature and is categorised as morphological or physiological depending on the mechanism and speed of the change. Colour change has been assumed to carry physiological costs, but current knowledge has not advanced beyond this basic assumption. The costs of changing colour will shape the evolution of colour change in animals, yet no coherent research has been conducted in this area, leaving a gap in our understanding. Therefore, in this Review, we examine the direct and indirect evidence of the physiological cost of colour change from the cellular to the population level, in animals that utilise chromatophores in colour change. Our Review concludes that the physiological costs result from either one or a combination of the processes of (i) production, (ii) translocation and (iii) maintenance of pigments within the colour-containing cells (chromatophores). In addition, both types of colour change (morphological and physiological) pose costs as they require energy for hormone production and neural signalling. Moreover, our Review upholds the hypothesis that, if repetitively used, rapid colour change (i.e. seconds-minutes) is more costly than slow colour change (days-weeks) given that rapidly colour-changing animals show mitigations, such as avoiding colour change when possible. We discuss the potential implications of this cost on colour change, behaviour and evolution of colour-changing animals, generating testable hypotheses and emphasising the need for future work to address this gap.

Cephalopod-Inspired Design of Photomechanically Modulated Display Systems for On-Demand Fluorescent Patterning.

Cephalopods can display variable body color/patterns upon environmental stimulation via bioelectricity-controlled muscle contraction/expansion of skin chromatophores. However, it remains challenging to produce artificial display analogs that exhibit reversible and rapid switching between multiple expected luminescent patterns, although such systems are very appealing for many practical uses (e.g., data encryption). Inspired by the bioelectromechanical display tactic of cephalopods, in this work, a conceptually new photomechanically modulated fluorescent system that enables on-demand display of fluorescent patterns via a cascading stimulation-mechanical movement-optical output conduction mechanism is presented. Specifically, this biomimetic system comprises a customizable hollow display panel and a bottom-tethered photothermally responsive fluorescent actuator. Under NIR light, the photomechanically bending movements of the fluorescent actuator will immediately cover the hollow window of the display panel and synchronously manifest as the display of fluorescent patterns. Owing to its desirable time- and light-power-dependent actuating behaviors, diverse fluorescent patterns/information can be dynamically and reversibly displayed by facilely controlling this single remote NIR signal. This bioinspired strategy is universal and promising for fabricating on-demand fluorescent display platforms that combine a wide choice of fluorophores, remote control with high spatial/temporal precision, and especially single-input multiple-output features.

Visualization of Sox10-positive chromatoblasts by GFP fluorescence in flounder larvae and juveniles using electroporation.

Japanese flounder are left-right asymmetrical, with features, such as dark, ocular-side specific pigmentation. This pigmentation arises during metamorphic stages, along with the asymmetric differentiation of adult-type chromatophores. Additionally, among juveniles, tank-reared specimens commonly show ectopic pigmentation on their blind sides. In both cases, neural crest-derived Sox10-positive progenitor cells at the dorsal fin base are hypothesized to contribute to chromatophore development. Here, we developed a method to visualize Sox10-positive cells via green fluorescent protein (GFP) fluorescence to directly monitor their migration and differentiation into chromatophores in vivo. Electroporation was applied to introduce GFP reporter vectors into the dorsal fin base of larvae and juveniles. Cre-loxP system vectors were also tested to enable cell labeling even after a decrease in sox10 expression levels. In larvae, undifferentiated Sox10-positive progenitor cells were labeled in the dorsal fin base, whereas newly differentiated adult-type chromatophores were seen dispersed on the ocular side. In juveniles, Sox10-positive cells were identified in the connective tissue of the dorsal fin base and observed prominently in areas of ectopic pigmentation, including several labeled melanophores. Thus, it was suggested that during metamorphic stages, Sox10-positive cells at the dorsal fin base contribute to adult-type chromatophore development, whereas in juveniles, they persist as precursors in the connective tissue, which in response to stimuli migrate to generate ectopic pigmentation. These findings contribute to elucidating pigmentation mechanisms, as well as abnormalities seen in hatchery-reared flounders. The electroporation method may be adapted to diverse animals as an accessible gene transfer method in various research fields, including developmental and biomedical studies.

In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning.

Skin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish's color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.

Highly Efficient Knockout of a Squid Pigmentation Gene.

Seminal studies using squid as a model led to breakthroughs in neurobiology. The squid giant axon and synapse, for example, laid the foundation for our current understanding of the action potential [1], ionic gradients across cells [2], voltage-dependent ion channels [3], molecular motors [4-7], and synaptic transmission [8-11]. Despite their anatomical advantages, the use of squid as a model receded over the past several decades as investigators turned to genetically tractable systems. Recently, however, two key advances have made it possible to develop techniques for the genetic manipulation of squid. The first is the CRISPR-Cas9 system for targeted gene disruption, a largely species-agnostic method [12, 13]. The second is the sequencing of genomes for several cephalopod species [14-16]. If made genetically tractable, squid and other cephalopods offer a wealth of biological novelties that could spur discovery. Within invertebrates, not only do they possess by far the largest brains, they also express the most sophisticated behaviors [17]. In this paper, we demonstrate efficient gene knockout in the squid Doryteuthis pealeii using CRISPR-Cas9. Ommochromes, the pigments found in squid retinas and chromatophores, are derivatives of tryptophan, and the first committed step in their synthesis is normally catalyzed by Tryptophan 2,3 Dioxygenase (TDO [18-20]). Knocking out TDO in squid embryos efficiently eliminated pigmentation. By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled. Genotyping revealed knockout efficiencies routinely greater than 90%. This study represents a critical advancement toward making squid genetically tractable.

A quantitative modelling approach to zebrafish pigment pattern formation.

Pattern formation is a key aspect of development. Adult zebrafish exhibit a striking striped pattern generated through the self-organisation of three different chromatophores. Numerous investigations have revealed a multitude of individual cell-cell interactions important for this self-organisation, but it has remained unclear whether these known biological rules were sufficient to explain pattern formation. To test this, we present an individual-based mathematical model incorporating all the important cell-types and known interactions. The model qualitatively and quantitatively reproduces wild type and mutant pigment pattern development. We use it to resolve a number of outstanding biological uncertainties, including the roles of domain growth and the initial iridophore stripe, and to generate hypotheses about the functions of leopard. We conclude that our rule-set is sufficient to recapitulate wild-type and mutant patterns. Our work now leads the way for further in silico exploration of the developmental and evolutionary implications of this pigment patterning system.

Bioluminescent backlighting illuminates the complex visual signals of a social squid in the deep sea.

Visual signals rapidly relay information, facilitating behaviors and ecological interactions that shape ecosystems. However, most known signaling systems can be restricted by low light levels-a pervasive condition in the deep ocean, the largest inhabitable space on the planet. Resident visually cued animals have therefore been hypothesized to have simple signals with limited information-carrying capacity. We used cameras mounted on remotely operated vehicles to study the behavior of the Humboldt squid, Dosidicus gigas, in its natural deep-sea habitat. We show that specific pigmentation patterns from its diverse repertoire are selectively displayed during foraging and in social scenarios, and we investigate how these behaviors may be used syntactically for communication. We additionally identify the probable mechanism by which D. gigas, and related squids, illuminate these patterns to create visual signals that can be readily perceived in the deep, dark ocean. Numerous small subcutaneous (s.c.) photophores (bioluminescent organs) embedded throughout the muscle tissue make the entire body glow, thereby backlighting the pigmentation patterns. Equipped with a mechanism by which complex information can be rapidly relayed through a visual pathway under low-light conditions, our data suggest that the visual signals displayed by D. gigas could share design features with advanced forms of animal communication. Visual signaling by deep-living cephalopods will likely be critical in understanding how, and how much, information can be shared in one of the planet's most challenging environments for visual communication.

Pigmentation formation and expression analysis of tyrosinase in Siniperca chuatsi.

Animal pigmentation primarily depends on the presence and mixing ratio of chromatophores, functioning in animal survival and communication. For the benthic and carnivorous Siniperca chuatsi, pigmentation pattern is key to concealment and predation. In this study, the formation, distribution, and main pattern of chromatophores were observed in the embryos, larvae, skins, and visceral tissues from S. chuatsi. Melanophores were firstly visualized in the yolk sac at segmentation stage, and then they were migrated to the whole body and further clustered into the black stripes, bands, and patches. In adult S. chuatsi, the head, black band, and body side skins mainly contained melanophores, showing as deep or light black. The abdomen skin mainly contained iridophores, showing as silvery. In the eye, the pigment layers were located in the epithelial layers of iris and retina and shown as black. Then, the pigmentation-related gene, tyrosinase gene from S. chuatsi (Sc-tyr) was analyzed by bioinformatics and quantitative methods. The Sc-tyr gene encoded a protein with 540 amino acids (Sc-TYR). The Sc-TYR contained two copper ion binding sites, which were coordinated by six conserved histidines (H182, H205, H214, H366, H370, H393) and necessary for catalytic activity. The Sc-TYR was well conserved compared with TYR of various species with higher degree of sequence similarity with other fishes (77.6-98.3%). The qRT-PCR test showed that the Sc-tyr mRNA reached the peak value at segmentation stage in the embryo development, the black skins displayed a higher expression level than that in silvery skin, and the eye had the highest expression level compared with other tissues. Further research on enzyme activity showed that the expression patterns of tyrosinase activity were similar to that of the Sc-tyr mRNA. Comparing with the results of molecular and phenotype, it was found that the temporal and spatial distributions of tyrosinase corresponded well with changes in pigmentation patterns and the intensity of skin melanization. This study initially explored the pigmentation formation and tyrosinase expression, which served as a foundation for further insight into the genetics mechanism of body color formation in S. chuatsi.

Extraocular Vision in a Brittle Star Is Mediated by Chromatophore Movement in Response to Ambient Light.

Almost all animals can sense light, but only those with spatial vision can "see." Conventionally, this was restricted to animals possessing discrete visual organs (eyes), but extraocular vision could facilitate vision without eyes. Echinoderms form the focus of extraocular vision research [1-7], and the brittle star Ophiocoma wendtii, which exhibits light-responsive color change and shelter seeking, became a key species of interest [4, 8, 9]. Both O. wendtii and an apparently light-indifferent congeneric, O. pumila, possess an extensive network of r-opsin-reactive cells, but its function remains unclear [4]. We show that, although both species are strongly light averse, O. wendtii orients to stimuli necessitating spatial vision for detection, but O. pumila does not. However, O. wendtii's response disappears when chromatophores are contracted within the skeleton. Combining immunohistochemistry, histology, and synchrotron microtomography, we reconstructed models of photoreceptors in situ and extracted estimated angular apertures for O. wendtii and O. pumila. Angular sensitivity estimates, derived from these models, support the hypothesis that chromatophores constitute a screening mechanism in O. wendtii, providing sufficient resolving power to detect the stimuli. RNA sequencing (RNA-seq) identified opsin candidates in both species, including multiple r-opsins and transduction pathway constituents, congruent with immunohistochemistry and studies of other echinoderms [10, 11]. Finally, we note that differing body postures between the two species during experiments may reflect aspect of signal integration. This represents one of the most detailed mechanisms for extraocular vision yet proposed and draws interesting parallels with the only other confirmed extraocular visual system, that of some sea urchins, which also possess chromatophores [1].

Microscale light management and inherent optical properties of intact corals studied with optical coherence tomography.

Coral reefs are highly productive photosynthetic systems and coral optics studies suggest that such high efficiency is due to optimized light scattering by coral tissue and skeleton. Here, we characterize the inherent optical properties, i.e. the scattering coefficient, µs, and the anisotropy of scattering, g, of eight intact coral species using optical coherence tomography (OCT). Specifically, we describe light scattering by coral skeletons, coenoarc tissues, polyp tentacles and areas covered by fluorescent pigments (FP). Our results reveal that light scattering between coral species ranges from µs = 3 mm-1 ( Stylophora pistillata) to µs = 25 mm-1 ( Echinopora lamelosa) . For Platygyra pini, µs was 10-fold higher for tissue versus skeleton, while in other corals (e.g. Hydnophora pilosa) no difference was found between tissue and skeletal scattering. Tissue scattering was threefold enhanced in coenosarc tissues ( µs = 24.6 mm-1) versus polyp tentacles ( µs = 8.3 mm-1) in Turbinaria reniformis. FP scattering was almost isotropic when FP were organized in granule chromatophores ( g = 0.34) but was forward directed when FP were distributed diffusely in the tissue ( g = 0.96). Our study provides detailed measurements of coral scattering and establishes a rapid approach for characterizing optical properties of photosynthetic soft tissues via OCT in vivo.

Cyclic nature of the REM sleep-like state in the cuttlefish Sepia officinalis.

Sleep is a state of immobility characterized by three key criteria: an increased threshold of arousal, rapid reversal to an alert state and evidence of homeostatic 'rebound sleep' in which there is an increase in the time spent in this quiescent state following sleep deprivation. Common European cuttlefish, Sepia officinalis, show states of quiescence during which they meet the last two of these three criteria, yet also show spontaneous bursts of arm and eye movements that accompany rapid changes in chromatophore patterns in the skin. Here, we report that this rapid eye movement sleep-like (REMS-like) state is cyclic in nature. Iterations of the REMS-like state last 2.42±0.22 min (mean±s.e.m.) and alternate with 34.01±1.49 min of the quiescent sleep-like state for durations lasting 176.89±36.71 min. We found clear evidence that this REMS-like state (i) occurs in animals younger than previously reported; (ii) follows an ultradian pattern; (iii) includes intermittent dynamic chromatophore patterning, representing fragments of normal patterning seen in the waking state for a wide range of signaling and camouflage; and (iv) shows variability in the intensity of expression of these skin patterns between and within individuals. These data suggest that cephalopods, which are mollusks with an elaborate brain and complex behavior, possess a sleep-like state that resembles behaviorally the vertebrate REM sleep state, although the exact nature and mechanism of this form of sleep may differ from that of vertebrates.

Elucidating the control and development of skin patterning in cuttlefish.

Few animals provide a readout that is as objective of their perceptual state as camouflaging cephalopods. Their skin display system includes an extensive array of pigment cells (chromatophores), each expandable by radial muscles controlled by motor neurons. If one could track the individual expansion states of the chromatophores, one would obtain a quantitative description-and potentially even a neural description by proxy-of the perceptual state of the animal in real time. Here we present the use of computational and analytical methods to achieve this in behaving animals, quantifying the states of tens of thousands of chromatophores at sixty frames per second, at single-cell resolution, and over weeks. We infer a statistical hierarchy of motor control, reveal an underlying low-dimensional structure to pattern dynamics and uncover rules that govern the development of skin patterns. This approach provides an objective description of complex perceptual behaviour, and a powerful means to uncover the organizational principles that underlie the function, dynamics and morphogenesis of neural systems.

Evolution of Endothelin signaling and diversification of adult pigment pattern in Danio fishes.

Fishes of the genus Danio exhibit diverse pigment patterns that serve as useful models for understanding the genes and cell behaviors underlying the evolution of adult form. Among these species, zebrafish D. rerio exhibit several dark stripes of melanophores with sparse iridophores that alternate with light interstripes of dense iridophores and xanthophores. By contrast, the closely related species D. nigrofasciatus has an attenuated pattern with fewer melanophores, stripes and interstripes. Here we demonstrate species differences in iridophore development that presage the fully formed patterns. Using genetic and transgenic approaches we identify the secreted peptide Endothelin-3 (Edn3)-a known melanogenic factor of tetrapods-as contributing to reduced iridophore proliferation and fewer stripes and interstripes in D. nigrofasciatus. We further show the locus encoding this factor is expressed at lower levels in D. nigrofasciatus owing to cis-regulatory differences between species. Finally, we show that functions of two paralogous loci encoding Edn3 have been partitioned between skin and non-skin iridophores. Our findings reveal genetic and cellular mechanisms contributing to pattern differences between these species and suggest a model for evolutionary changes in Edn3 requirements for pigment patterning and its diversification across vertebrates.

Primary Endosymbiosis: Emergence of the Primary Chloroplast and the Chromatophore, Two Independent Events.

The emergence of semiautonomous organelles, such as the mitochondrion, the chloroplast, and more recently, the chromatophore, are critical steps in the evolution of eukaryotes. They resulted from primary endosymbiotic events that seem to share general features, i.e., an acquisition of a bacterium/cyanobacteria likely via a phagocytic membrane, a genome reduction coinciding with an escape of genes from the organelle to the nucleus, and finally the appearance of an active system translocating nuclear-encoded proteins back to the organelles. An intense mobilization of foreign genes of bacterial origin, via horizontal gene transfers, plays a critical role. Some third partners, like Chlamydia, might have facilitated the transition from cyanobacteria to the early chloroplast. This chapter describes our current understanding of primary endosymbiosis, with a specific focus on primary chloroplasts considered to have emerged more than one billion years ago, and on the chromatophore, having emerged about one hundred million years ago.

An introduction to color-changing systems from the cephalopod protein reflectin.

Cephalopods possess unrivaled camouflage and signaling abilities that are enabled by their sophisticated skin, wherein multiple layers contain chromatophore pigment cells (as part of larger chromatophore organs) and different types of reflective cells called iridocytes and leucophores. The optical functionality of these cells (and thus cephalopod skin) critically relies upon subcellular structures partially composed of unusual structural proteins known as reflectins. Herein, we highlight studies that have investigated reflectins as materials within the context of color-changing coatings. We in turn discuss these proteins' multi-faceted properties, associated challenges, and future potential. Through our presentation of selected case studies, we hope to stimulate additional dialogue and spur further research on photonic technologies based on and inspired by reflectins.

Myogenic activity and serotonergic inhibition in the chromatophore network of the squid Dosidicus gigas (family Ommastrephidae) and Doryteuthis opalescens (family Loliginidae).

Seemingly chaotic waves of spontaneous chromatophore activity occur in the ommastrephid squid Dosidicus gigas in the living state and immediately after surgical disruption of all known inputs from the central nervous system. Similar activity is apparent in the loliginid Doryteuthis opalescens, but only after chronic denervation of chromatophores for 5-7 days. Electrically stimulated, neurally driven activity in intact individuals of both species is blocked by tetrodotoxin (TTX), but TTX has no effect on spontaneous wave activity in either D. gigas or denervated D. opalescens Spontaneous TTX-resistant activity of this sort is therefore likely myogenic, and such activity is eliminated in both preparations by serotonin (5-HT), a known inhibitor of chromatophore activity. Immunohistochemical techniques reveal that individual axons containing L-glutamate or 5-HT (and possibly both in a minority of processes) are associated with radial muscle fibers of chromatophores in intact individuals of both species, although the area of contact between both types of axons and muscle fibers is much smaller in D. gigas Glutamatergic and serotonergic axons degenerate completely following denervation in D. opalescens Spontaneous waves of chromatophore activity in both species are thus associated with reduced (or no) serotonergic input in comparison to the situation in intact D. opalescens Such differences in the level of serotonergic inhibition are consistent with natural chromogenic behaviors in these species. Our findings also suggest that such activity might propagate via the branching distal ends of radial muscle fibers.