Molecular Basis of Chemotactile Sensation in Octopus.

Animals display wide-ranging evolutionary adaptations based on their ecological niche. Octopuses explore the seafloor with their flexible arms using a specialized "taste by touch" system to locally sense and respond to prey-derived chemicals and movement. How the peripherally distributed octopus nervous system mediates relatively autonomous arm behavior is unknown. Here, we report that octopus arms use a family of cephalopod-specific chemotactile receptors (CRs) to detect poorly soluble natural products, thereby defining a form of contact-dependent, aquatic chemosensation. CRs form discrete ion channel complexes that mediate the detection of diverse stimuli and transduction of specific ionic signals. Furthermore, distinct chemo- and mechanosensory cells exhibit specific receptor expression and electrical activities to support peripheral information coding and complex chemotactile behaviors. These findings demonstrate that the peripherally distributed octopus nervous system is a key site for signal processing and highlight how molecular and anatomical features synergistically evolve to suit an animal's environmental context.

Wake-like skin patterning and neural activity during octopus sleep.

While sleeping, many vertebrate groups alternate between at least two sleep stages: rapid eye movement and slow wave sleep1-4, in part characterized by wake-like and synchronous brain activity, respectively. Here we delineate neural and behavioural correlates of two stages of sleep in octopuses, marine invertebrates that evolutionarily diverged from vertebrates roughly 550 million years ago (ref. 5) and have independently evolved large brains and behavioural sophistication. 'Quiet' sleep in octopuses is rhythmically interrupted by approximately 60-s bouts of pronounced body movements and rapid changes in skin patterning and texture6. We show that these bouts are homeostatically regulated, rapidly reversible and come with increased arousal threshold, representing a distinct 'active' sleep stage. Computational analysis of active sleep skin patterning reveals diverse dynamics through a set of patterns conserved across octopuses and strongly resembling those seen while awake. High-density electrophysiological recordings from the central brain reveal that the local field potential (LFP) activity during active sleep resembles that of waking. LFP activity differs across brain regions, with the strongest activity during active sleep seen in the superior frontal and vertical lobes, anatomically connected regions associated with learning and memory function7-10. During quiet sleep, these regions are relatively silent but generate LFP oscillations resembling mammalian sleep spindles11,12 in frequency and duration. The range of similarities with vertebrates indicates that aspects of two-stage sleep in octopuses may represent convergent features of complex cognition.

Structural basis of sensory receptor evolution in octopus.

Chemotactile receptors (CRs) are a cephalopod-specific innovation that allow octopuses to explore the seafloor via 'taste by touch'1. CRs diverged from nicotinic acetylcholine receptors to mediate contact-dependent chemosensation of insoluble molecules that do not readily diffuse in marine environments. Here we exploit octopus CRs to probe the structural basis of sensory receptor evolution. We present the cryo-electron microscopy structure of an octopus CR and compare it with nicotinic receptors to determine features that enable environmental sensation versus neurotransmission. Evolutionary, structural and biophysical analyses show that the channel architecture involved in cation permeation and signal transduction is conserved. By contrast, the orthosteric ligand-binding site is subject to diversifying selection, thereby mediating the detection of new molecules. Serendipitous findings in the cryo-electron microscopy structure reveal that the octopus CR ligand-binding pocket is exceptionally hydrophobic, enabling sensation of greasy compounds versus the small polar molecules detected by canonical neurotransmitter receptors. These discoveries provide a structural framework for understanding connections between evolutionary adaptations at the atomic level and the emergence of new organismal behaviour.

Sensory specializations drive octopus and squid behaviour.

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated 'taste by touch' sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs1 and nicotinic receptors2. These analyses demonstrate an evolutionary transition from an ancestral aromatic 'cage' that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour.

Topology, dynamics, and control of a muscle-architected soft arm.

Muscular hydrostats, such as octopus arms or elephant trunks, lack bones entirely, endowing them with exceptional dexterity and reconfigurability. Key to their unmatched ability to control nearly infinite degrees of freedom is the architecture into which muscle fibers are weaved. Their arrangement is, effectively, the instantiation of a sophisticated mechanical program that mediates, and likely facilitates, the control and realization of complex, dynamic morphological reconfigurations. Here, by combining medical imaging, biomechanical data, live behavioral experiments, and numerical simulations, an octopus-inspired arm made of [Formula: see text]200 continuous muscle groups is synthesized, exposing "mechanically intelligent" design and control principles broadly pertinent to dynamics and robotics. Such principles are mathematically understood in terms of storage, transport, and conversion of topological quantities, effected into complex 3D motions via simple muscle activation templates. These are in turn composed into higher-level control strategies that, compounded by the arm's compliance, are demonstrated across challenging manipulation tasks, revealing surprising simplicity and robustness.

Evidence for tactile 3D shape discrimination by octopus.

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.

Passive elasticity properties of Octopus rubescens arms.

In this report, passive elasticity properties of Octopus rubescens arm tissue are investigated using a multidisciplinary approach encompassing biomechanical experiments, computational modeling, and analyses. Tensile tests are conducted to obtain stress-strain relationships of the arm under axial stretch. Rheological tests are also performed to probe the dynamic shear response of the arm tissue. Based on these tests, comparisons against three different viscoelasticity models are reported.

Biomechanics, motor control and dynamic models of the soft limbs of the octopus and other cephalopods.

Muscular hydrostats are organs composed entirely of packed arrays of incompressible muscles and lacking any skeletal support. Found in both vertebrates and invertebrates, they are of great interest for comparative biomechanics from engineering and evolutionary perspectives. The arms of cephalopods (e.g. octopus and squid) are particularly interesting muscular hydrostats because of their flexibility and ability to generate complex behaviors exploiting elaborate nervous systems. Several lines of evidence from octopus studies point to the use of both brain and arm-embedded motor control strategies that have evolved to simplify the complexities associated with the control of flexible and hyper-redundant limbs and bodies. Here, we review earlier and more recent experimental studies on octopus arm biomechanics and neural motor control. We review several dynamic models used to predict the kinematic characteristics of several basic motion primitives, noting the shortcomings of the current models in accounting for behavioral observations. We also discuss the significance of impedance (stiffness and viscosity) in controlling the octopus's motor behavior. These factors are considered in light of several new models of muscle biomechanics that could be used in future research to gain a better understanding of motor control in the octopus. There is also a need for updated models that encompass stiffness and viscosity for designing and controlling soft robotic arms. The field of soft robotics has boomed over the past 15 years and would benefit significantly from further progress in biomechanical and motor control studies on octopus and other muscular hydrostats.

Contact chemoreception in multi-modal sensing of prey by Octopus.

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.

Behavioral changes in senescent giant Pacific octopus (Enteroctopus dofleini) are associated with peripheral neural degeneration and loss of epithelial tissue.

Most species of octopus experience extreme physical decline after a single reproductive bout which extends over a period of days, weeks, or months before eventual death. Although outward indicators of senescence are widely recognized, comparatively little is known about physiological and neural changes accompanying terminal decline in octopuses. Here, we measured changes in behavioral response to nociceptive stimuli across the lifespan in giant Pacific octopus (GPO), Enteroctopus dofleini, held in public aquariums in the USA. Post-euthanasia, tissue was collected from arm tips, and neural and epithelial cell degeneration was quantified and compared with biopsies of arm tips from healthy, pre-reproductive GPOs. Behavioral assays showed significant changes both in low threshold mechanosensory responses and nociceptive behavioral responses beginning early in senescence and extending until euthanasia. Histology data showed that while the ratio of apoptotic cells to total cell number stayed constant between healthy and senescent GPOs, overall neural and epithelial cell density was significantly lower in terminally senescent octopuses compared with healthy controls. Our data provide new insight into the time-course and causes of sensory dysfunction in senescent cephalopods and suggest proactive welfare management should begin early in the senescence phase, well before animals enter terminal decline.

Plain-Body Octopus's (Callistoctopus aspilosomatis) Learning about Objects via Both Visual and Tactile Sensory Inputs: A Pilot Study.

Although various recognizing abilities have been revealed for octopuses, they predominantly deal with only a few species. Therefore, cognition diversity among other octopus species that have been overlooked needs to be investigated. We investigated whether plain-body octopus can learn a symbolic stimulus, for the reason that this octopus is abundant around Okinawa Island with a complex coral community landscape. Attention was paid to whether an octopus can learn a stimulus based solely on visual information without previous experience of learning it tactilely as well as visually. Furthermore, we examined whether different sensory inputs affect learning in octopuses. First, we tested whether octopuses can be conditioned to three different stimuli (object, picture, and video of a white cross). Octopuses that were presented an object or a picture could learn to touch them. However, octopuses that were presented a video could not learn to touch the stimulus. Second, we showed a video to octopuses that had already learned about an object or a picture to investigate whether the octopuses, having experienced a target using visual and tactile senses, can recognize a video of the target based solely on visual information. Octopuses could learn to touch the video. When a conditioned stimulus and a novel stimulus were simultaneously presented on a computer screen, an octopus that had learned an object more often selected the conditioned stimulus when compared with an octopus that had experienced only a picture. These findings suggest that octopuses use multisensory information to recognize a specific object.

Exploring the effects of warming seas by using the optimal and pejus temperatures of the embryo of three Octopoda species in the Gulf of Mexico.

Using data related to thermal optimal and pejus of the embryos of Octopus americanus from Brazil and O. insularis and O. maya from Mexico, this study aimed to project the potential distribution areas in the Gulf of Mexico and predict distribution shifts under different Representative Concentration Pathway scenarios (RCP 6 and 8.5) for the years 2050 and 2100. The different thermal tolerances elicited different responses to current and future scenarios. In this sense, O. insularis and O. maya thermal niches stretch from the Caribbean to Florida. Nevertheless, O. insularis may inhabit warmer areas than O. maya. Surprisingly, no area was considered thermally habitable for O. americanus, which could have been associated with the use of data of populations thermally adapted to temperate conditions south of Brazil. According to models, a warming scenario would cause a restriction of the available thermal niche of O. maya, while O. insularis could expand under RCP 6 scenarios. This restriction was more substantial in the RCP 8.5 scenario. Nevertheless, under the RCP 8.5 scenario, the temperature in 2100 may negatively affect even O. insularis, the species most thermal tolerant. If our results are accurate, the fishing yield of O. insularis will increase in the future, replacing the heavily exploited O. maya in the coasts of the southern Gulf of Mexico. Regarding O. americanus, no inference might be made until thermal tolerances of locally adapted populations can be studied.

From synaptic input to muscle contraction: arm muscle cells of Octopus vulgaris show unique neuromuscular junction and excitation-contraction coupling properties.

The muscular-hydrostat configuration of octopus arms allows high manoeuvrability together with the efficient motor performance necessary for its multitasking abilities. To control this flexible and hyper-redundant system the octopus has evolved unique strategies at the various levels of its brain-to-body organization. We focus here on the arm neuromuscular junction (NMJ) and excitation-contraction (E-C) properties of the arm muscle cells. We show that muscle cells are cholinergically innervated at single eye-shaped locations where acetylcholine receptors (AChR) are concentrated, resembling the vertebrate neuromuscular endplates. Na+ and K+ contribute nearly equally to the ACh-activated synaptic current mediating membrane depolarization, thereby activating voltage-dependent L-type Ca2+ channels. We show that cell contraction can be mediated directly by the inward Ca2+ current and also indirectly by calcium-induced calcium release (CICR) from internal stores. Indeed, caffeine-induced cell contraction and immunohistochemical staining revealed the presence and close association of dihydropyridine (DHPR) and ryanodine (RyR) receptor complexes, which probably mediate the CICR. We suggest that the dynamics of octopus arm contraction can be controlled in two ways; motoneurons with large synaptic inputs activate vigorous contraction via activation of the two routs of Ca2+ induced contraction, while motoneurons with lower-amplitude inputs may regulate a graded contraction through frequency-dependent summation of EPSP trains that recruit the CICR. Our results thus suggest that these motoneuronal pools are likely to be involved in the activation of different E-C coupling modes, thus enabling a dynamics of muscles activation appropriate for various tasks such as stiffening versus motion generation.

From injury to full repair: nerve regeneration and functional recovery in the common octopus, Octopus vulgaris.

Spontaneous nerve regeneration in cephalopod molluscs occurs in a relative short time after injury, achieving functional recovery of lost capacity. In particular, transection of the pallial nerve in the common octopus (Octopus vulgaris) determines the loss and subsequent restoration of two functions fundamental for survival, i.e. breathing and skin patterning, the latter involved in communication between animals and concealment. The phenomena occurring after lesion have been investigated in a series of previous studies, but a complete analysis of the changes taking place at the level of the axons and the effects on the animals' appearance during the whole regenerative process is still missing. Our goal was to determine the course of events following injury, from impairment to full recovery. Through imaging of the traced damaged nerves, we were able to characterize the pathways followed by fibres during regeneration and end-target re-innervation, while electrophysiology and behavioural observations highlighted the regaining of functional connections between the central brain and periphery, using the contralateral nerve in the same animal as an internal control. The final architecture of a fully regenerated pallial nerve does not exactly mirror the original structure; however, functionality returns to match the phenotype of an intact octopus with no observable impact on the behaviour of the animal. Our findings provide new important scenarios for the study of regeneration in cephalopods and highlight the octopus pallial nerve as a valuable 'model' among invertebrates.

Vision is highly sensitive to oxygen availability in marine invertebrate larvae.

For many animals, evolution has selected for complex visual systems despite the high energetic demands associated with maintaining eyes and their processing structures. Therefore, the metabolic demands of visual systems make them highly sensitive to fluctuations in available oxygen. In the marine environment, oxygen changes over daily, seasonal and inter-annual time scales, and there are large gradients of oxygen with depth. Vision is linked to survival in many marine animals, particularly among the crustaceans, cephalopods and fish, and early life stages of these groups rely on vision for prey capture, predator detection and their distribution in the water column. Using in vivo electroretinogram recordings, we show that there is a decrease in retinal sensitivity to light in marine invertebrates when exposed to reduced oxygen availability. We found a 60-100% reduction in retinal responses in the larvae of cephalopods and crustaceans: the market squid (Doryteuthis opalescens), the two-spot octopus (Octopus bimaculatus), the tuna crab (Pleuroncodes planipes) and the graceful rock crab (Metacarcinus gracilis). A decline in oxygen also decreases the temporal resolution of vision in D. opalescens These results are the first demonstration that vision in marine invertebrates is highly sensitive to oxygen availability and that the thresholds for visual impairment from reduced oxygen are species-specific. Oxygen-impaired retinal function may change the visual behaviors crucial to survival in these marine larvae. These findings may impact our understanding of species' vulnerability to ocean oxygen loss and suggest that researchers conducting electrophysiology experiments should monitor oxygen levels, as even small changes in oxygen may affect the results.

Multiple optic gland signaling pathways implicated in octopus maternal behaviors and death.

Post-reproductive life in the female octopus is characterized by an extreme pattern of maternal care: the mother cares for her clutch of eggs without feeding until her death. These maternal behaviors are eradicated if the optic glands, the octopus analog of the vertebrate pituitary gland, are removed from brooding females. Despite the optic gland's importance in regulating maternal behavior, the molecular features underlying optic gland function are unknown. Here, we identify major signaling systems of the Octopus bimaculoides optic gland. Through behavioral analyses and transcriptome sequencing, we report that the optic gland undergoes remarkable molecular changes that coincide with transitions between behavioral stages. These include the dramatic upregulation and downregulation of catecholamine, steroid, insulin and feeding peptide pathways. Transcriptome analyses in other tissues demonstrate that these molecular changes are not generalized markers of senescence, but instead, specific features of the optic glands. Our study expands the classic optic gland-pituitary gland analogy and more specifically, it indicates that, rather than a single 'self-destruct' hormone, the maternal optic glands employ multiple pathways as systemic hormonal signals of behavioral regulation.