Evolutionary history influences the microbiomes of a female symbiotic reproductive organ in cephalopods.

Many female squids and cuttlefishes have a symbiotic reproductive organ called the accessory nidamental gland (ANG) that hosts a bacterial consortium involved with egg defense against pathogens and fouling organisms. While the ANG is found in multiple cephalopod families, little is known about the global microbial diversity of these ANG bacterial symbionts. We used 16S rRNA gene community analysis to characterize the ANG microbiome from different cephalopod species and assess the relationship between host and symbiont phylogenies. The ANG microbiome of 11 species of cephalopods from four families (superorder: Decapodiformes) that span seven geographic locations was characterized. Bacteria of class Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were found in all species, yet analysis of amplicon sequence variants by multiple distance metrics revealed a significant difference between ANG microbiomes of cephalopod families (weighted/unweighted UniFrac, Bray-Curtis, P = 0.001). Despite being collected from widely disparate geographic locations, members of the family Sepiolidae (bobtail squid) shared many bacterial taxa including (~50%) Opitutae (Verrucomicrobia) and Ruegeria (Alphaproteobacteria) species. Furthermore, we tested for phylosymbiosis and found a positive correlation between host phylogenetic distance and bacterial community dissimilarity (Mantel test r = 0.7). These data suggest that closely related sepiolids select for distinct symbionts from similar bacterial taxa. Overall, the ANGs of different cephalopod species harbor distinct microbiomes and thus offer a diverse symbiont community to explore antimicrobial activity and other functional roles in host fitness.IMPORTANCEMany aquatic organisms recruit microbial symbionts from the environment that provide a variety of functions, including defense from pathogens. Some female cephalopods (squids, bobtail squids, and cuttlefish) have a reproductive organ called the accessory nidamental gland (ANG) that contains a bacterial consortium that protects eggs from pathogens. Despite the wide distribution of these cephalopods, whether they share similar microbiomes is unknown. Here, we studied the microbial diversity of the ANG in 11 species of cephalopods distributed over a broad geographic range and representing 15-120 million years of host divergence. The ANG microbiomes shared some bacterial taxa, but each cephalopod species had unique symbiotic members. Additionally, analysis of host-symbiont phylogenies suggests that the evolutionary histories of the partners have been important in shaping the ANG microbiome. This study advances our knowledge of cephalopod-bacteria relationships and provides a foundation to explore defensive symbionts in other systems.

Effect of visual lateralization on the spatial position of individuals within a school of oval squid (Sepioteuthis lessoniana).

The spatial position of individuals within a social group, which provides the group members with benefits and costs, is determined by several physical and physiological factors. Lateralization (left and right asymmetry of morphology and behavior) could also be factors determining the individual's positions within a group. However, this possibility has been documented in some fish species, but never in an invertebrate species. This study investigates the association between spatial positions and lateralization in oval squid, Sepioteuthis lessoniana, which displays social behavior, such as schooling and lateralization for eye use (visual lateralization). The direction and strength of visual lateralization were determined for single squid by observing which eye was used to detect the prey, predators, and conspecifics. The spatial positions of individuals were determined by identifying whether the squids were in the left or right side from the center of the school. When the prey was presented to schooling squids, strongly lateralized squids against prey positioned themselves on the right side, whereas weakly lateralized squids positioned themselves on the left side. When the predator was presented to squids, the strongly lateralized squids against the conspecifics positioned themselves on the right side, and the weakly lateralized squids positioned themselves on the left side. When no targets were presented, the strongly lateralized squids against the predator positioned themselves on the right side, whereas the weakly lateralized squids positioned themselves on the left side. The strength of visual lateralization of oval squid could offer the defensive and offensive functions of schools with specific individual positions.

Evaluation of Visual and Tactile Perception by Plain-Body Octopus (Callistoctopus aspilosomatis) of Prey-Like Objects.

We investigated the characteristic features of perception in octopuses by examining multisensory information from an object simulating prey, which provided different visual and tactile stimuli. In experiments, we presented plain-body octopus with four kinds of models, namely, the Lifelike crab, the Embedded crab, the Translucent crab, and the Black cuboid. These models contain different amounts of visual and tactile information that a crab originally contains: the Lifelike crab resembles a crab both visually and tactilely, the Embedded crab resembles a crab visually but provides different tactile information, the Translucent crab provides tactile information of a crab but contains less visual information, and the Black cuboid lacks both visual and tactile information of a crab. Among these four models, octopuses contacted most with the Lifelike crab, which was similar to their behavior with a crab. Indeed, octopuses were fastest to contact the Lifelike crab and had the longest duration of contacting it among the four models. Octopuses contacted the Embedded crab more than the Translucent crab, both of which had contrasting visuo-tactile information compared to that of a crab. Quickness of octopuses to contact and duration of contact with the Embedded crab were more similar to those with the Lifelike crab than to those with the Translucent crab. Furthermore, octopuses contacted the Black cuboid least among the models. These results suggest that octopuses compositely detect both visual and tactile information in order to perceive an object. Furthermore, octopuses possess the potential priority either for visual or tactile information, by which they process the target object.

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.

Tropical Octopus Abdopus aculeatus Can Learn to Recognize Real and Virtual Symbolic Objects.

We used three consecutive operant conditioning tasks to determine whether the tropical octopus Abdopus aculeatus is able to learn to recognize a symbolic object, in either real or virtual forms. In Experiment 1, we examined whether octopuses can be conditioned to a real object (a white ball) and whether such trained individuals can select the conditioned object when they are presented with an unconditioned object. We show that octopuses learned to respond to and select the conditioned white ball in preference to the unconditioned object. In Experiment 2, we examined whether octopuses can be conditioned to an object that gradually changes from real to virtual (i.e., an image of that object on a computer screen). We presented four types of objects, all variations of a white ball, in a stepwise sequence as a conditioned stimulus: a real white ball, a real image of a white ball without a margin, a real image of a white ball centered within a black margin, and a virtual image of a white ball (a video on a computer screen). Individual octopuses learned to respond to all three real objects, and then a subset of these octopuses responded to the virtual object. In Experiment 3, we examined whether an octopus can learn a virtual image of an object with a specific shape not tested in Experiments 1 and 2. We presented octopuses with an image of a white cross, which was placed at various distances (i.e., close, medium, and far). We found that after having learned these images, octopuses could learn the virtual white cross on a computer screen. Furthermore, when we simultaneously presented octopuses with a conditioned virtual object and an unconditioned virtual object, they selected the former. Through these three experiments, we confirmed that A. aculeatus can learn both real and virtual specific objects.

Development of a contrast-enhanced micro computed tomography protocol for the oval squid (Sepioteuthis lessoniana) brain.

Coleoid cephalopods (squid, cuttlefish, and octopus) have a well-developed and complex central nervous system. Its absolute size is the largest among invertebrates, and the brain-to-body mass ratio is larger than that of fish and reptiles and equivalent to that of birds and mammals. Although a number of histological studies have been conducted on the brains of cephalopods, most of them used a light microscope or an electron microscope, which show the microstructure of the brain, but often cannot image the whole brain instantaneously. Of late, micro computed tomography (CT) has gained popularity for imaging animal brains because it allows for noninvasive three-dimensional (3D) reconstruction and preprocessing that are not cumbersome. To perform micro-CT on cephalopod brains, we first tested conditions suitable for preprocessing, paying special attention to staining conditions that would provide high contrast images. Four agents, iodine in 99.5% ethanol, iodine potassium iodide in water (IKI), phosphotungstic acid in 70% ethanol, and nonionic iodinated contrast agent in water, were tested at various concentrations and durations on brain of juvenile oval squid. To evaluate the quality of staining, we calculated the contrast ratio of the two-dimensional (2D) images and compared 3D segmentation of the best and worst 2D images. We concluded that 3% IKI staining for 7 days was the best combination to enhance the images contrast of the oval squid brain, in which each brain lobe was clearly detected and 3D segmentation of the whole brain was possible. The wider applicability of this preprocessing method for micro-CT of the brains of other cephalopods is discussed.

Visual Stimuli for the Induction of Hunting Behavior in Cuttlefish Sepia pharaonis.

Cuttlefish exhibit typical hunting behavior, including elongating tentacles against specific prey such as prawn and mysid shrimp. Cuttlefish hunting behavior involves three different actions: attention, positioning, and seizure. Hunting behavior is innate and stereotypic behavior, and it is present in newly hatched juveniles. Factors associated with prey are known to induce this behavior, similar to the sign stimulus, whereby young herring chicks imitate pecking behavior against a red dot on their parent's bill. Although the hunting behavior of cuttlefish has been described and used as an indicator to test learning and memory, details of a stimulus that can elicit this behavior remain unknown. Here, we used a variety of visual stimuli presented on a computer screen to investigate the factors that induce hunting behavior of pharaoh cuttlefish, Sepia pharaonis. We found that the appearance of prey (western king prawn, Melicertus latisulcatus) and their movement at a vertical angle of 45° are specific factors that can initiate hunting behavior. We also showed that the height of prey can attract cuttlefish and initiate hunting. To the best of our knowledge, this is the first report of a stimulus that elicits stereotyped hunting behavior by coleoid cephalopods.

Environmental enrichment affects the ontogeny of learning, memory, and depth perception of the pharaoh cuttlefish Sepia pharaonis.

We investigated the effects of environmental enrichment on the cognitive abilities of pharaoh cuttlefish, Sepia pharaonis, which were reared from day seven in four different environments: isolated, poor, standard, and enriched. First, we used "prawn-in-the-tube" to test whether environmental enrichment affects the ontogeny of learning and memory of S. pharaonis. The results showed that cuttlefish could usually learn the task regardless of their age and environment. At early age (74 - 81 d), cuttlefish from the isolated environment memorized the task for 24h. However, at later age (104 - 171 d), the isolated cuttlefish were unable to remember the task. In addition, cuttlefish from the poor environment could not memorize the task at all ages examined. Cuttlefish from the standard environment could memorize the task in later ages (134 - 171 d). In contrast, cuttlefish from the enriched environment could memorize the task at all ages examined. Second, we examined the effect of environmental enrichment on the ontogeny of depth perception of S. pharaonis by observing their hunting behavior. Distance from the prey during hunting was always greater in isolated cuttlefish than those from the other three environments. In addition, hunting success and number of prey captured were always lowest in the isolated cuttlefish for all ages. In contrast, hunting success was always the highest in cuttlefish from the enriched environment. These variations in behavior among cuttlefish raised in different environments suggest that the visual/tactic input derived from social and physical factors of the surrounding environment could promote maturation of learning, memory, and depth perception in S. pharaonis.

Correction to: Unique arm-flapping behavior of the pharaoh cuttlefish, Sepia pharaonis: putative mimicry of a hermit crab.

[This corrects the article DOI: 10.1007/s10164-017-0519-7.].

Unique arm-flapping behavior of the pharaoh cuttlefish, Sepia pharaonis: putative mimicry of a hermit crab.

Cephalopods are able to control their arms sophisticatedly and use them for various behaviors, such as camouflage, startling predators and hunting prey. Here, we report a previously undescribed arm-flapping behavior of the pharaoh cuttlefish, Sepia pharaonis, observed in captivity. S. pharaonis raised the first pair of arms and wrinkled the parts near the distal end, where the skin color was darkened. Additionally, S. pharaonis spread the second and third pairs of arms and bent them as if they were jointed, and flapped the distal ends. S. pharaonis showed this behavior in two different situations: after being introduced into a large space, and during hunting. We discuss the putative functions of this behavior, including possible mimicry of a hermit crab, considering the situations in which the behavior was observed.

Environmental Enrichment Accelerates the Ontogeny of Cryptic Behavior in Pharaoh Cuttlefish (Sepia pharaonis).

We examined effect of environmental enrichment on cuttlefish, the most neutrally advanced invertebrate, to compare species variation of genetic and environmental influences. Cuttlefish were reared from seven to 117 days in one of three environments, namely, "poor" (artificial bottom without objects), "standard" (sandy bottom), and "enriched" (sandy bottom with objects). In Experiment 1, we explored whether enrichment affects the exhibition of crypsis in the cuttlefish. The cuttlefish in the standard and enriched environments spent most of their time at the bottom, exhibiting the mottled or disruptive pattern starting at 27 days of age. On the contrary, those in the poor environment exhibited uniform pattern starting at 87 days of age. Additionally, they repeatedly attempted to dig from 27 to 87 days of age, and moved around by hovering from 77 to 117 days of age. In Experiment 2, we exposed the cuttlefish to six novel substrates every other month after 53 days of age to verify whether enrichment actually affected the maturation of cryptic ability. Cuttlefish from the poor environment tended not to dig into white sandy bottom at 53-55 days of age. Additionally, they did not clearly exhibit appropriate body patterns in response to the six substrates compared to those from the other two environments at 81-83 days of age. However, at 113-115 days of age, most cuttlefish from the three environments exhibited similar cryptic behaviors in response to novel substrates. We conclude that physical enrichment promotes crypsis and accelerates the maturation of this ability in cuttlefish.

Effects of Visual Cues of a Moving Model Predator on Body Patterns in Cuttlefish Sepia pharaonis.

We examined the effects of predator-prey distance (PPD) and trajectory of the predator on the body patterns that the pharaoh cuttlefish, Sepia pharaonis, shows in response to a predator. A model predator moving in three different trajectories was presented to the cuttlefish: T1, approached the cuttlefish but bypassed above; T2, approached directly toward the cuttlefish; T3, bypassed the cuttlefish both vertically and horizontally. We divided the body patterns that the cuttlefish expressed into seven categories, i.e., "uniform light", "disruptive", "center circle", "dark square", "vertical stripe", "all dark" and "eyespots". In T1, the number of individuals that showed "dark square" increased as the model approached the cuttlefish, whereas the number of individuals that showed "disruptive" decreased. In T2, the number of individuals that showed "all dark" and "eyespots" increased as the model approached the cuttlefish. In T3, the number of individuals that showed "dark square" and "vertical stripe" increased as the model approached the cuttlefish, and it tended to decrease as the model receded from the cuttlefish. These results demonstrate that S. pharaonis changes its body patterns according to PPD and the trajectory of the predator, which would affect predation risk and/or predator perception.

Comparison of the ontogeny of hunting behavior in pharaoh cuttlefish (Sepia pharaonis) and oval squid (Sepioteuthis lessoniana).

Animals adopt various forms of hunting according to their ecological, morphological, and cognitive features, and their specific hunting skills are acquired ontogenetically in relation to these features. It is noted that cuttlefish and squid hunt prey through the elongation of tentacles used specifically to capture prey. However, these two cephalopods have different lifestyles, leading to questions such as whether hunting skill is acquired similarly after birth and whether tentacle elongation is behaviorally identical. To address these questions, we observed and compared how captive pharaoh cuttlefish (Sepia pharaonis) and oval squid (Sepioteuthis lessoniana) attack prey during their early life stages. Like the adults, S. pharaonis hatchlings used the tentacular lunge attack, whereas S. lessoniana hatchlings used the arm-opening attack. S. lessoniana began to exhibit the tentacular strike attack after 30 days of age. In addition to timing of the emergence of a specific hunting mode, some differences were observed in the physical aspect of hunting behavior. For cuttlefish, maximum tentacle length and maximum speed of tentacle elongation increased from hatching to 30 days of age and then decreased. In contrast, for squid, maximum tentacle length increased from hatching to 30 days of age and then became constant. The distance to prey was positively correlated with maximum length and speed of tentacle elongation in S. pharaonis and with maximum swimming speed in S. lessoniana. These results show that cuttlefish mainly use an ambush strategy and that squid use a pursuit strategy. Possible causes for the ontogenetic differences in hunting behavior are discussed.