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Cephalopods produce dynamic colors and skin patterns for communication and camouflage via stratified networks of neuronally actuated yellow, red, and brown chromatophore organs, each filled with thousands of pigment granules. While compositional analysis of chromatophore granules in Doryteuthis pealeii reveals the pigments as ommochromes, the ultrastructural features of the granules and their effects on bulk coloration have not been explored. To investigate this, we isolated granules from specific colored chromatophores and imaged them using multiple modalities. The brown granules are largest with smooth surface coatings. Red granules are intermediate in size with irregular surface textures, and yellow granules are smallest, with rough, porous surfaces. Many of the granules contain sub-granular features that also vary in presentation with color. Correlated light and electron microscopy reveal that differences in hue of individual granules are similarly associated with size, shape, and texture, suggesting that granules may be structurally adapted to modify the dominant visible colors presented within the chromatophores. These findings suggest that granule ultrastructure, not just chemical composition, may be significant in producing the range of colors presented in cephalopod chromatophores., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Octopuses integrate visual, chemical and tactile sensory information while foraging and feeding in complex marine habitats. The respective roles of these modes are of interest ecologically, neurobiologically, and for development of engineered soft robotic arms. While vision guides their foraging path, benthic octopuses primarily search "blindly" with their arms to find visually hidden prey amidst rocks, crevices and coral heads. Each octopus arm is lined with hundreds of suckers that possess a combination of chemo- and mechanoreceptors to distinguish prey. Contact chemoreception has been demonstrated in lab tests, but mechanotactile sensing is less well characterized. We designed a non-invasive live animal behavioral assay that isolated mechanosensory capabilities of Octopus bimaculoides arms and suckers to discriminate among five resin 3D-printed prey and non-prey shapes (all with identical chemical signatures). Each shape was introduced inside a rock dome and was only accessible to the octopus' arms. Octopuses' responses were variable. Young octopuses discriminated the crab prey shape from the control, whereas older octopuses did not. These experiments suggest that mechanotactile sensing of 3D shapes may aid in prey discrimination; however, (i) chemo-tactile information may be prioritized over mechanotactile information in prey discrimination, and (ii) mechanosensory capability may decline with age., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Deimatic behaviours, also referred to as startle behaviours, are used against predators and rivals. Although many are spectacular, their proximate and ultimate causes remain unclear. In this review we aim to synthesise what is known about deimatic behaviour and identify knowledge gaps. We propose a working hypothesis for deimatic behaviour, and discuss the available evidence for the evolution, ontogeny, causation, and survival value of deimatic behaviour using Tinbergen's Four Questions as a framework. Our overarching aim is to direct future research by suggesting ways to address the most pressing questions in this field., (© 2022 Cambridge Philosophical Society.)

Cuttlefish are active carnivores that possess a wide repertoire of body patterns that can be changed within milliseconds for many types of camouflage and communication. The forms and functions of many body patterns are well known from ethological studies in the field and laboratory. Yet one aspect has not been reported in detail: the category of rapid, brief and high-contrast changes in body coloration ("Tentacle Shot Patterns" or TSPs) that always occur with the ejection of two ballistic tentacles to strike live moving prey ("Tentacles Go Ballistic" or TGB moment). We designed and tested a mechanical device that presented prey in a controlled manner, taking advantage of a key stimulus for feeding: motion of the prey. High-speed video recordings show a rapid transition into TSPs starting 114 ms before TGB ( N = 114). TSPs are then suppressed as early as 470-500 ms after TGB ( P < 0.05) in unsuccessful hunts, while persisting for at least 3 s after TGB in successful hunts. A granularity analysis revealed significant differences in the large-scale high-contrast body patterning present in TSPs compared to the camouflage body pattern deployed beforehand. TSPs best fit the category of secondary defense called deimatic displaying, meant to briefly startle predators and interrupt their attack sequence while cuttlefish are distracted by striking prey. We characterize TSPs as a pattern category for which the main distinguishing feature is a high-contrast signaling pattern with aspects of Acute Conflict Mottle or Acute Disruptive Pattern. The data and methodology presented here open opportunities for quantifying the rapid neural responses in this visual sensorimotor set of behaviors., (© 2013-2022 The Journal of Biological Methods, All rights reserved.)

Octopuses have keen vision and are generally considered visual predators, yet octopuses predominantly forage blindly in nature, inserting their arms into crevices to search and detect hidden prey. The extent to which octopuses discriminate prey using chemo- versus mechano-tactile sensing is unknown. We developed a whole-animal behavioral assay that takes advantage of octopuses' natural searching behavior to test their ability to discriminate prey from non-prey tastes solely via contact chemoreception. This methodology eliminated vision, mechano-tactile sensing and distance chemoreception while testing the contact chemosensory discriminatory abilities of the octopus arm suckers. Extracts from two types of prey (crab, shrimp) and three types of non-prey (sea star, algae, seawater) were embedded in agarose (to control for mechano-tactile discrimination) and presented to octopuses inside an artificial rock dome; octopuses reached their arms inside to explore its contents - imitating natural prey-searching behavior. Results revealed that octopuses are capable of discriminating between potential prey items using only contact chemoreception, as measured by an increased amount of sucker contact time and arm curls when presented with prey extracts versus non-prey extracts. These results highlight the importance of contact chemoreception in the multi-modal sensing involved in a complex foraging behavior., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cephalopods are important in biologic and biomedical research, yet relatively little objective information is available to guide researchers and veterinarians regarding the best methods for anesthetizing these animals for various experimental procedures. Recent studies demonstrate that ethyl alcohol and magnesium chloride are effective at depressing efferent and afferent neural signals in some tropical cephalopod species when measured via the pallial nerve. Here we used similar methods to test 2 temperate species ( Octopus bimaculoides and Sepia officinalis ) and demonstrate that (1) ethyl alcohol and magnesium chloride were effective at reversibly depressing evoked activity in the pallial nerve, (2) ethyl alcohol generally had shorter induction and recovery times compared with magnesium chloride, (3) both agents were associated with a latency between the behavioral and neural effects, and it was longer with magnesium chloride, and (4) senescent animals generally had longer induction or recovery times than young animals. Both agents successfully anesthetized both life stages; however, our data show that assessing anesthesia based solely on behavior may lead to premature commencement of invasive procedures. We conclude that temperate cephalopods can be humanely, effectively, and completely anesthetized by using these 2 agents and that the loss of neural signal we show here is consistent with true anesthesia and not merely paralysis. This relatively simple, nondestructive nerve recording technique can be applied to the study of other prospective anesthetic agents in cephalopods.

Episodic memory, remembering past experiences based on unique what-where-when components, declines during ageing in humans, as does episodic-like memory in non-human mammals. By contrast, semantic memory, remembering learnt knowledge without recalling unique what-where-when features, remains relatively intact with advancing age. The age-related decline in episodic memory likely stems from the deteriorating function of the hippocampus in the brain. Whether episodic memory can deteriorate with age in species that lack a hippocampus is unknown. Cuttlefish are molluscs that lack a hippocampus. We test both semantic-like and episodic-like memory in sub-adults and aged-adults nearing senescence ( n = 6 per cohort). In the semantic-like memory task, cuttlefish had to learn that the location of a food resource was dependent on the time of day. Performance, measured as proportion of correct trials, was comparable across age groups. In the episodic-like memory task, cuttlefish had to solve a foraging task by retrieving what-where-when information about a past event with unique spatio-temporal features. In this task, performance was comparable across age groups; however, aged-adults reached the success criterion (8/10 correct choices in consecutive trials) significantly faster than sub-adults. Contrary to other animals, episodic-like memory is preserved in aged cuttlefish, suggesting that memory deterioration is delayed in this species.

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.", (© 2021 Wiley Periodicals LLC.)

The ability to exert self-control varies within and across taxa. Some species can exert self-control for several seconds whereas others, such as large-brained vertebrates, can tolerate delays of up to several minutes. Advanced self-control has been linked to better performance in cognitive tasks and has been hypothesized to evolve in response to specific socio-ecological pressures. These pressures are difficult to uncouple because previously studied species face similar socio-ecological challenges. Here, we investigate self-control and learning performance in cuttlefish, an invertebrate that is thought to have evolved under partially different pressures to previously studied vertebrates. To test self-control, cuttlefish were presented with a delay maintenance task, which measures an individual's ability to forgo immediate gratification and sustain a delay for a better but delayed reward. Cuttlefish maintained delay durations for up to 50-130 s. To test learning performance, we used a reversal-learning task, whereby cuttlefish were required to learn to associate the reward with one of two stimuli and then subsequently learn to associate the reward with the alternative stimulus. Cuttlefish that delayed gratification for longer had better learning performance. Our results demonstrate that cuttlefish can tolerate delays to obtain food of higher quality comparable to that of some large-brained vertebrates.

The octopus arm is often referred to as one of the most flexible limbs in nature, yet this assumption requires detailed inspection given that this has not been measured comprehensively for all portions of each arm. We investigated the diversity of arm deformations in Octopus bimaculoides with a frame-by-frame observational analysis of laboratory video footage in which animals were challenged with different tasks. Diverse movements in these hydrostatic arms are produced by some combination of four basic deformations: bending (orally, aborally; inward, outward), torsion (clockwise, counter-clockwise), elongation, and shortening. More than 16,500 arm deformations were observed in 120 min of video. Results showed that all eight arms were capable of all four types of deformation along their lengths and in all directions. Arms function primarily to bring the sucker-lined oral surface in contact with target surfaces. Bending was the most common deformation observed, although the proximal third of the arms performed relatively less bending and more shortening and elongation as compared with other arm regions. These findings demonstrate the exceptional flexibility of the octopus arm and provide a basis for investigating motor control of the entire arm, which may aid the future development of soft robotics.

Loliginid squids provide a unique model system to explore male alternative reproductive tactics (ARTs) and their linkage to size, behavioral decision making, and possibly age. Large individuals fight one another and the winners form temporary consortships with females, while smaller individuals do not engage in male-male agonistic bouts but use various sneaker tactics to obtain matings, each with varying mating and fertilization success. There is substantial behavioral flexibility in most species, as smaller males can facultatively switch to the alternative consort behaviors as the behavioral context changes. These forms of ARTs can involve different: mating posture; site of spermatophore deposition; fertilization success; and sperm traits. Most of the traits of male dimorphism (both anatomical and behavioral) are consistent with traditional sexual selection theory, while others have unique features that may have evolved in response to the fertilization environment faced by each temporary or permanent male morph., (Copyright © 2019 Marian, Apostólico, Chiao, Hanlon, Hirohashi, Iwata, Mather, Sato and Shaw.)

The European common cuttlefish, Sepia officinalis, is used extensively in biological and biomedical research, yet its microbiome remains poorly characterized. We analyzed the microbiota of the digestive tract, gills, and skin in mariculture-raised S. officinalis using a combination of 16S rRNA amplicon sequencing, quantitative PCR (qPCR), and fluorescence spectral imaging. Sequencing revealed a highly simplified microbiota consisting largely of two single bacterial amplicon sequence variants (ASVs) of Vibrionaceae and Piscirickettsiaceae . The esophagus was dominated by a single ASV of the genus Vibrio . Imaging revealed bacteria in the family Vibrionaceae distributed in a discrete layer that lines the esophagus. This Vibrio was also the primary ASV found in the microbiota of the stomach, cecum, and intestine, but occurred at lower abundance, as determined by qPCR, and was found only scattered in the lumen rather than in a discrete layer via imaging analysis. Treatment of animals with the commonly used antibiotic enrofloxacin led to a nearly 80% reduction of the dominant Vibrio ASV in the esophagus but did not significantly alter the relative abundance of bacteria overall between treated versus control animals. Data from the gills were dominated by a single ASV in the family Piscirickettsiaceae , which imaging visualized as small clusters of cells. We conclude that bacteria belonging to the Gammaproteobacteria are the major symbionts of the cuttlefish Sepia officinalis cultured from eggs in captivity and that the esophagus and gills are major colonization sites. IMPORTANCE Microbes can play critical roles in the physiology of their animal hosts, as evidenced in cephalopods by the role of Vibrio ( Aliivibrio ) fischeri in the light organ of the bobtail squid and the role of Alpha - and Gammaproteobacteria in the reproductive system and egg defense in a variety of cephalopods. We sampled the cuttlefish microbiome throughout the digestive tract, gills, and skin and found dense colonization of an unexpected site, the esophagus, by a microbe of the genus Vibrio , as well as colonization of gills by Piscirickettsiaceae . This finding expands the range of organisms and body sites known to be associated with Vibrio and is of potential significance for understanding host-symbiont associations, as well as for understanding and maintaining the health of cephalopods in mariculture.

Behavioural lateralization is widespread. Yet, a fundamental question remains, how can lateralization be evolutionary stable when individuals lateralized in one direction often significantly outnumber individuals lateralized in the opposite direction? A recently developed game theory model predicts that fitness consequences which occur during intraspecific interactions may be driving population-level lateralization as an evolutionary stable strategy. This model predicts that: (i) minority-type individuals exist because they are more likely to adopt unpredictable fighting behaviours during competitive interactions (e.g. fighting); and (ii) majority-type individuals exist because there is a fitness advantage in having their biases synchronized with other conspecifics during interactions that require coordination (e.g. mating). We tested these predictions by investigating biases in giant Australian cuttlefish during fighting and mating interactions. During fighting, most male cuttlefish favoured the left eye and these males showed higher contest escalation; but minority-type individuals with a right-eye bias achieved higher fighting success. During mating interactions, most male cuttlefish favoured the left eye to inspect females. Furthermore, most male cuttlefish approached the female's right side during a mating attempt and these males achieved higher mating success. Our data support the hypothesis that population-level biases are an evolutionary consequence of the fitness advantages involved in intraspecific interactions.

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.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

This study investigated how cuttlefish (Sepia officinalis) camouflage patterns are influenced by the proportions of different gray-scales present in visually cluttered environments. All experimental substrates comprised spatially random arrays of texture elements (texels) of five gray-scales: Black, Dark gray, Gray, Light gray, and White. The substrates in Experiment 1 were densely packed arrays of square texels that varied over 4 sizes in different conditions. Experiment 2 used substrates in which texels were disks separated on a homogeneous background that was Black, Gray or White in different conditions. In a given condition, the histogram of texel gray-scales was varied across different substrates. For each of 16 cuttlefish pattern response statistics c, the resulting data were used to determine the strength with which variations in the proportions of different gray-scales influenced c. The main finding is that darker-than-average texels (i.e., texels of negative contrast polarity) predominate in controlling cuttlefish pattern responses in the context of cluttered substrates. In Experiment 1, for example, substrates of all four texel-sizes, activation of the cuttlefish "white square" and "white head bar" (two highly salient skin components) is strongly influenced by variations in the proportions of Black and Dark gray (but not Gray, Light gray, or White) texels. It is hypothesized that in the context of high-variance visual input characteristic of cluttered substrates in the cuttlefish natural habitat, elements of negative contrast polarity reliably signal the presence of edges produced by overlapping objects, in the presence of which disruptive pattern responses are likely to achieve effective camouflage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

Technologies that use stretchable materials are increasingly important, yet we are unable to control how they stretch with much more sophistication than inflating balloons. Nature, however, demonstrates remarkable control of stretchable surfaces; for example, cephalopods can project hierarchical structures from their skin in milliseconds for a wide range of textural camouflage. Inspired by cephalopod muscular morphology, we developed synthetic tissue groupings that allowed programmable transformation of two-dimensional (2D) stretchable surfaces into target 3D shapes. The synthetic tissue groupings consisted of elastomeric membranes embedded with inextensible textile mesh that inflated to within 10% of their target shapes by using a simple fabrication method and modeling approach. These stretchable surfaces transform from flat sheets to 3D textures that imitate natural stone and plant shapes and camouflage into their background environments., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Male cuttlefish compete for females with a repertoire of visually dramatic behaviors. Laboratory experiments have explored this system in Sepia officinalis, but corroborative field data have eluded collection attempts by many researchers. While scuba diving in Turkey, we fortuitously filmed an intense sequence of consort/intruder behaviors in which the consort lost and then regained his female mate from the intruder. These agonistic bouts escalated in stages, leading to fast dramatic expression of the elaborate intense zebra display and culminating in biting and inking as the intruder male attempted a forced copulation of the female. When analyzed in the context of game theory, the patterns of fighting behavior were more consistent with mutual assessment than self-assessment of fighting ability. Additional observations of these behaviors in nature are needed to conclusively determine which models best represent conflict resolution, but our field observations agree with laboratory findings and provide a valuable perspective.

Masquerade is a defence tactic in which a prey resembles an inedible or inanimate object thus causing predators to misclassify it. Most masquerade colour patterns are static although some species adopt postures or behaviours to enhance the effect. Dynamic masquerade in which the colour pattern can be changed is rare. Here we report a two-step sensory process that enables an additional novel capability known only in cuttlefish and octopus: morphing three-dimensional physical skin texture that further enhances the optical illusions created by coloured skin patterns. Our experimental design incorporated sequential sensory processes: addition of a three-dimensional rock to the testing arena, which attracted the cuttlefish to settle next to it; then visual processing by the cuttlefish of physical textures on the rock to guide expression of the skin papillae, which can range from fully relaxed (smooth skin) to fully expressed (bumpy skin). When a uniformly white smooth rock was presented, cuttlefish moved to the rock and deployed a uniform body pattern with mostly smooth skin. When a rock with small-scale fragments of contrasting shells was presented, the cuttlefish deployed mottled body patterns with strong papillae expression. These robust and reversible responses indicate a sophisticated visual sensorimotor system for dynamic masquerade., (© 2017 The Author(s).)

Vertebrates with laterally placed eyes typically exhibit preferential eye use for ecological activities such as scanning for predators or prey. Processing visual information predominately through the left or right visual field has been associated with specialized function of the left and right brain. Lateralized vertebrates often share a general pattern of lateralized brain function at the population level, whereby the left hemisphere controls routine behaviors and the right hemisphere controls emergency responses. Recent studies have shown evidence of preferential eye use in some invertebrates, but whether the visual fields are predominately associated with specific ecological activities remains untested. We used the European common cuttlefish, Sepia officinalis , to investigate whether the visual field they use is the same, or different, during anti-predatory, and predatory behavior. To test for lateralization of anti-predatory behavior, individual cuttlefish were placed in a new environment with opaque walls, thereby obliging them to choose which eye to orient away from the opaque wall to scan for potential predators (i.e., vigilant scanning). To test for lateralization of predatory behavior, individual cuttlefish were placed in the apex of an isosceles triangular arena and presented with two shrimp in opposite vertexes, thus requiring the cuttlefish to choose between attacking a prey item to the left or to the right of them. Cuttlefish were significantly more likely to favor the left visual field to scan for potential predators and the right visual field for prey attack. Moreover, individual cuttlefish that were leftward directed for vigilant scanning were predominately rightward directed for prey attack. Lateralized individuals also showed faster decision-making when presented with prey simultaneously. Cuttlefish appear to have opposite directions of lateralization for anti-predatory and predatory behavior, suggesting that there is functional specialization of each optic lobe (i.e., brain structures implicated in visual processing). These results are discussed in relation to the role of lateralized brain function and the evolution of population level lateralization.

Animals with detectable injuries are at escalated threat of predation. The anti-predation tactic of schooling reduces individual predation risk overall, but it is not known how schooling behavior affects injured animals, or whether risks are reduced equally for injured animals versus other school members. In this laboratory study we examined the effects of minor fin injury on schooling decisions made by squid. Schooling behavior of groups of squid, in which one member was injured, was monitored over 24h. Injured squid were more likely to be members of a school shortly after injury (0.5-2h), but there were no differences compared with sham-injured squid at longer time points (6-24h). Overall, the presence of an injured conspecific increased the probability that a school would form, irrespective of whether the injured squid was a member of the school. When groups containing one injured squid were exposed to a predator cue, injured squid were more likely to join the school, but their position depended on whether the threat was a proximate visual cue or olfactory cue. We found no evidence that injured squid oriented themselves to conceal their injury from salient threats. Overall we conclude that nociceptive sensitization after injury changes grouping behaviors in ways that are likely to be adaptive., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Sound is a widely available cue in aquatic environments and is used by many marine animals for vital behaviors. Most research has focused on marine vertebrates. Relatively little is known about sound detection in marine invertebrates despite their abundance and importance in marine environments. Cephalopods are a key taxon in many ecosystems, but their behavioral interactions relative to acoustic stimuli have seldom been studied. Here we review current knowledge regarding (1) the frequency ranges and sound levels that generate behavioral responses and (2) the types of behavioral responses and their biological relevance.

The slender filefish is a master of adaptive camouflage and can change its appearance within 1-3 seconds. Videos and photographs of this animal's cryptic body patterning and behavior were collected in situ under natural light on a Caribbean coral reef. We present an ethogram of body patterning components that includes large- and small-scale spots, stripes and bars that confer a variety of cryptic patterns amidst a range of complex backgrounds. Field images were analyzed to investigate two aspects of camouflage effectiveness: (i) the degree of color resemblance between animals and their nearby visual stimuli and (ii) the visibility of each fish's actual body outline versus its illusory outline. Most animals more closely matched the color of nearby visual stimuli than that of the surrounding background. Three-dimensional dermal flaps complement the melanophore skin patterns by enhancing the complexity of the fish's physical skin texture to disguise its actual body shape, and the morphology of these structures was studied. The results suggest that the body patterns, skin texture, postures and swimming orientations putatively hinder both the detection and recognition of the fish by potential visual predators. Overall, the rapid speed of change of multiple patterns, color blending with nearby backgrounds, and the physically complicated edge produced by dermal flaps effectively camouflage this animal among soft corals and macroalgae in the Caribbean Sea.

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., (© 2015 Marine Biological Laboratory.)

Cephalopods are famous for their ability to change color and pattern rapidly for signaling and camouflage. They have keen eyes and remarkable vision, made possible by photoreceptors in their retinas. External to the eyes, photoreceptors also exist in parolfactory vesicles and some light organs, where they function using a rhodopsin protein that is identical to that expressed in the retina. Furthermore, dermal chromatophore organs contain rhodopsin and other components of phototransduction (including retinochrome, a photoisomerase first found in the retina), suggesting that they are photoreceptive. In this study, we used a modified whole-mount immunohistochemical technique to explore rhodopsin and retinochrome expression in a number of tissues and organs in the longfin squid, Doryteuthis pealeii. We found that fin central muscles, hair cells (epithelial primary sensory neurons), arm axial ganglia, and sucker peduncle nerves all express rhodopsin and retinochrome proteins. Our findings indicate that these animals possess an unexpected diversity of extraocular photoreceptors and suggest that extraocular photoreception using visual opsins and visual phototransduction machinery is far more widespread throughout cephalopod tissues than previously recognized.

We review recent research on the visual mechanisms of rapid adaptive camouflage in cuttlefish. These neurophysiologically complex marine invertebrates can camouflage themselves against almost any background, yet their ability to quickly (0.5-2 s) alter their body patterns on different visual backgrounds poses a vexing challenge: how to pick the correct body pattern amongst their repertoire. The ability of cuttlefish to change appropriately requires a visual system that can rapidly assess complex visual scenes and produce the motor responses-the neurally controlled body patterns-that achieve camouflage. Using specifically designed visual backgrounds and assessing the corresponding body patterns quantitatively, we and others have uncovered several aspects of scene variation that are important in regulating cuttlefish patterning responses. These include spatial scale of background pattern, background intensity, background contrast, object edge properties, object contrast polarity, object depth, and the presence of 3D objects. Moreover, arm postures and skin papillae are also regulated visually for additional aspects of concealment. By integrating these visual cues, cuttlefish are able to rapidly select appropriate body patterns for concealment throughout diverse natural environments. This sensorimotor approach of studying cuttlefish camouflage thus provides unique insights into the mechanisms of visual perception in an invertebrate image-forming eye.

Cephalopod mollusks are renowned for their colorful and dynamic body patterns, produced by an assemblage of skin components that interact with light. These may include iridophores, leucophores, chromatophores and (in some species) photophores. Here, we present molecular evidence suggesting that cephalopod chromatophores - small dermal pigmentary organs that reflect various colors of light - are photosensitive. RT-PCR revealed the presence of transcripts encoding rhodopsin and retinochrome within the retinas and skin of the squid Doryteuthis pealeii, and the cuttlefish Sepia officinalis and Sepia latimanus. In D. pealeii, Gqα and squid TRP channel transcripts were present in the retina and in all dermal samples. Rhodopsin, retinochrome and Gqα transcripts were also found in RNA extracts from dissociated chromatophores isolated from D. pealeii dermal tissues. Immunohistochemical staining labeled rhodopsin, retinochrome and Gqα proteins in several chromatophore components, including pigment cell membranes, radial muscle fibers, and sheath cells. This is the first evidence that cephalopod dermal tissues, and specifically chromatophores, may possess the requisite combination of molecules required to respond to light., (© 2015. Published by The Company of Biologists Ltd.)

Sound is a widely available and vital cue in aquatic environments, yet most bioacoustic research has focused on marine vertebrates, leaving sound detection in invertebrates poorly understood. Cephalopods are an ecologically key taxon that likely use sound and may be impacted by increasing anthropogenic ocean noise, but little is known regarding their behavioral responses or adaptations to sound stimuli. These experiments identify the acoustic range and levels that elicit a wide range of secondary defense behaviors such as inking, jetting and rapid coloration change. Secondarily, it was found that cuttlefish habituate to certain sound stimuli. The present study examined the behavioral responses of 22 cuttlefish (Sepia officinalis) to pure-tone pips ranging from 80 to 1000 Hz with sound pressure levels of 85-188 dB re. 1 μPa rms and particle accelerations of 0-17.1 m s(-2). Cuttlefish escape responses (inking, jetting) were observed between frequencies of 80 and 300 Hz and at sound levels above 140 dB re. 1 μPa rms and 0.01 m s(-2) (0.74 m s(-2) for inking responses). Body patterning changes and fin movements were observed at all frequencies and sound levels. Response intensity was dependent upon stimulus amplitude and frequency, suggesting that cuttlefish also possess loudness perception with a maximum sensitivity around 150 Hz. Cuttlefish habituated to repeated 200 Hz tone pips, at two sound intensities. Total response inhibition was not reached, however, and a basal response remained present in most animals. The graded responses provide a loudness sensitivity curve and suggest an ecological function for sound use in cephalopods., (© 2014. Published by The Company of Biologists Ltd.)

This field study describes the camouflage pattern repertoire, associated behaviours and speed of pattern change of Nassau groupers Epinephelus striatus at Little Cayman Island, British West Indies. Three basic camouflaged body patterns were observed under natural conditions and characterized quantitatively. The mean speed of pattern change across the entire body was 4.44 s (range = 0.97-9.87 s); the fastest pattern change as well as contrast change within a fixed pattern occurred within 1 s. Aside from apparent defensive camouflage, E. striatus used camouflage offensively to approach crustacean or fish prey, and three successful predation events were recorded. Although animal camouflage is a widespread tactic, dynamic camouflage is relatively uncommon and has been studied rarely in marine teleosts under natural conditions. The rapid changes observed in E. striatus suggest direct neural control of some skin colouration elements, and comparative studies of functional morphology and behaviour of colour change in other coral-reef teleosts are likely to reveal new mechanisms and adaptations of dynamic colouration., (© 2014 The Fisheries Society of the British Isles.)

Octopus, squid, cuttlefish, and other cephalopods exhibit exceptional capabilities for visually adapting to or differentiating from the coloration and texture of their surroundings, for the purpose of concealment, communication, predation, and reproduction. Long-standing interest in and emerging understanding of the underlying ultrastructure, physiological control, and photonic interactions has recently led to efforts in the construction of artificial systems that have key attributes found in the skins of these organisms. Despite several promising options in active materials for mimicking biological color tuning, existing routes to integrated systems do not include critical capabilities in distributed sensing and actuation. Research described here represents progress in this direction, demonstrated through the construction, experimental study, and computational modeling of materials, device elements, and integration schemes for cephalopod-inspired flexible sheets that can autonomously sense and adapt to the coloration of their surroundings. These systems combine high-performance, multiplexed arrays of actuators and photodetectors in laminated, multilayer configurations on flexible substrates, with overlaid arrangements of pixelated, color-changing elements. The concepts provide realistic routes to thin sheets that can be conformally wrapped onto solid objects to modulate their visual appearance, with potential relevance to consumer, industrial, and military applications.

Sublethal injury triggers long-lasting sensitization of defensive responses in most species examined, suggesting the involvement of powerful evolutionary selection pressures [1]. In humans, this persistent nociceptive sensitization is often accompanied by heightened sensations of pain and anxiety [2]. While experimental [3] and clinical [4] evidence support the adaptive value of immediate nociception during injury, no direct evidence exists for adaptive benefits of long-lasting sensitization after injury. Recently, we showed that minor injury produces long-term sensitization of behavioral and neuronal responses in squid, Doryteuthis pealei [5, 6]. Here we tested the adaptive value of this sensitization during encounters between squid and a natural fish predator. Locomotion and other spontaneous behaviors of squid that received distal injury to a single arm (with or without transient anesthesia) showed no measurable impairment 6 hr after the injury. However, black sea bass given access to freely swimming squid oriented toward and pursued injured squid at greater distances than uninjured squid, regardless of previous anesthetic treatment. Once targeted, injured squid began defensive behavioral sequences [7, 8] earlier than uninjured squid. This effect was blocked by brief anesthetic treatment that prevented development of nociceptive sensitization [6, 9]. Importantly, the early anesthetic treatment also reduced the subsequent escape and survival of injured, but not uninjured, squid. Thus, while minor injury increases the risk of predatory attack, it also triggers a sensitized state that promotes enhanced responsiveness to threats, increasing the survival (Darwinian fitness) of injured animals during subsequent predatory encounters., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A major component of cephalopod adaptive camouflage behavior has rarely been studied: their ability to change the three-dimensionality of their skin by morphing their malleable dermal papillae. Recent work has established that simple, conical papillae in cuttlefish (Sepia officinalis) function as muscular hydrostats; that is, the muscles that extend a papilla also provide its structural support. We used brightfield and scanning electron microscopy to investigate and compare the functional morphology of nine types of papillae of different shapes, sizes and complexity in six species: S. officinalis small dorsal papillae, Octopus vulgaris small dorsal and ventral eye papillae, Macrotritopus defilippi dorsal eye papillae, Abdopus aculeatus major mantle papillae, O. bimaculoides arm, minor mantle, and dorsal eye papillae, and S. apama face ridge papillae. Most papillae have two sets of muscles responsible for extension: circular dermal erector muscles arranged in a concentric pattern to lift the papilla away from the body surface and horizontal dermal erector muscles to pull the papilla's perimeter toward its core and determine shape. A third set of muscles, retractors, appears to be responsible for pulling a papilla's apex down toward the body surface while stretching out its base. Connective tissue infiltrated with mucopolysaccharides assists with structural support. S. apama face ridge papillae are different: the contraction of erector muscles perpendicular to the ridge causes overlying tissues to buckle. In this case, mucopolysaccharide-rich connective tissue provides structural support. These six species possess changeable papillae that are diverse in size and shape, yet with one exception they share somewhat similar functional morphologies. Future research on papilla morphology, biomechanics and neural control in the many unexamined species of octopus and cuttlefish may uncover new principles of actuation in soft, flexible tissue.

Squid display impressive changes in body coloration that are afforded by two types of dynamic skin elements: structural iridophores (which produce iridescence) and pigmented chromatophores. Both color elements are neurally controlled, but nothing is known about the iridescence circuit, or the environmental cues, that elicit iridescence expression. To tackle this knowledge gap, we performed denervation, electrical stimulation and behavioral experiments using the long-fin squid, Doryteuthis pealeii. We show that while the pigmentary and iridescence circuits originate in the brain, they are wired differently in the periphery: (1) the iridescence signals are routed through a peripheral center called the stellate ganglion and (2) the iridescence motor neurons likely originate within this ganglion (as revealed by nerve fluorescence dye fills). Cutting the inputs to the stellate ganglion that descend from the brain shifts highly reflective iridophores into a transparent state. Taken together, these findings suggest that although brain commands are necessary for expression of iridescence, integration with peripheral information in the stellate ganglion could modulate the final output. We also demonstrate that squid change their iridescence brightness in response to environmental luminance; such changes are robust but slow (minutes to hours). The squid's ability to alter its iridescence levels may improve camouflage under different lighting intensities.

Commercial off-the-shelf digital cameras are inexpensive and easy-to-use instruments that can be used for quantitative scientific data acquisition if images are captured in raw format and processed so that they maintain a linear relationship with scene radiance. Here we describe the image-processing steps required for consistent data acquisition with color cameras. In addition, we present a method for scene-specific color calibration that increases the accuracy of color capture when a scene contains colors that are not well represented in the gamut of a standard color-calibration target. We demonstrate applications of the proposed methodology in the fields of biomedical engineering, artwork photography, perception science, marine biology, and underwater imaging.

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.

We evaluated cuttlefish (Sepia officinalis) responses to three teleost predators: bluefish (Pomatomus saltatrix), summer flounder (Paralichthys dentatus), and black seabass (Centropristis striata). We hypothesized that the distinct body shapes, swimming behaviors, and predation tactics exhibited by the three fishes would elicit markedly different antipredator responses by cuttlefish. Over the course of 25 predator-prey behavioral trials, 3 primary and 15 secondary defense behaviors of cuttlefish were shown to predators. In contrast, secondary defenses were not shown during control trials in which predators were absent. With seabass-a benthic, sit-and-pursue predator-cuttlefish used flight and spent more time swimming in the water column than with other predators. With bluefish-an active, pelagic searching predator-cuttlefish remained closely associated with the substrate and relied more on cryptic behaviors. Startle (deimatic) displays were the most frequent secondary defense shown to seabass and bluefish, particularly the Dark eye ring and Deimatic spot displays. We were unable to evaluate secondary defenses by cuttlefish to flounder-a lie-and-wait predator-because flounder did not pursue cuttlefish or make attacks. Nonetheless, cuttlefish used primary defense during flounder trials, alternating between cryptic still and moving behaviors. Overall, our results suggest that cuttlefish may vary their behavior in the presence of different teleost predators: cryptic behaviors may be more important in the presence of active searching predators (e.g., bluefish), while conspicuous movements such as swimming in the water column and startle displays may be more prevalent with relatively sedentary, bottom-associated predators (e.g., seabass).