Smart sharks: a review of chondrichthyan cognition.

450 million years of evolution have given chondrichthyans (sharks, rays and allies) ample time to adapt perfectly to their respective everyday life challenges and cognitive abilities have played an important part in that process. The diversity of niches that sharks and rays occupy corresponds to matching diversity in brains and behaviour, but we have only scratched the surface in terms of investigating cognition in this important group of animals. The handful of species that have been cognitively assessed in some detail over the last decade have provided enough data to safely conclude that sharks and rays are cognitively on par with most other vertebrates, including mammals and birds. Experiments in the lab as well as in the wild pose their own unique challenges, mainly due to the handling and maintenance of these animals as well as controlling environmental conditions and elimination of confounding factors. Nonetheless, significant advancements have been obtained in the fields of spatial and social cognition, discrimination learning, memory retention as well as several others. Most studies have focused on behaviour and the underlying neural substrates involved in cognitive information processing are still largely unknown. Our understanding of shark cognition has multiple practical benefits for welfare and conservation management but there are obvious gaps in our knowledge. Like most marine animals, sharks and rays face multiple threats. The effects of climate change, pollution and resulting ecosystem changes on the cognitive abilities of sharks and stingrays remain poorly investigated and we can only speculate what the likely impacts might be based on research on bony fishes. Lastly, sharks still suffer from their bad reputation as mindless killers and are heavily targeted by commercial fishing operations for their fins. This public relations issue clouds people's expectations of shark intelligence and is a serious impediment to their conservation. In the light of the fascinating results presented here, it seems obvious that the general perception of sharks and rays as well as their status as sentient, cognitive animals, needs to be urgently revisited.

Visual discrimination and resolution in freshwater stingrays (Potamotrygon motoro).

Potamotrygon motoro has been shown to use vision to orient in a laboratory setting and has been successfully trained in cognitive behavioral studies using visual stimuli. This study explores P. motoro's visual discrimination abilities in the context of two-alternative forced-choice experiments, with a focus on shape and contrast, stimulus orientation, and visual resolution. Results support that stingrays are able to discriminate stimulus-presence and -absence, overall stimulus contrasts, two forms, horizontal from vertical stimulus orientations, and different colors that also vary in brightness. Stingrays tested in visual resolution experiments demonstrated a range of visual acuities from < 0.13 to 0.23 cpd under the given experimental conditions. Additionally, this report includes the first evidence for memory retention in this species.

Not just shades of grey: life is full of colour for the ocellate river stingray (Potamotrygon motoro).

Previous studies have shown that marine stingrays have the anatomical and physiological basis for colour vision, with cone spectral sensitivity in the blue to green range of the visible spectrum. Behavioural studies on Glaucostegus typus also showed that blue and grey can be perceived and discriminated. The present study is the first to assess visual opsin genetics in the ocellate river stingray (Potamotrygon motoro) and test whether individuals perceive colour in two alternative forced choice experiments. Retinal transcriptome profiling using RNA-Seq and quantification demonstrated the presence of lws and rh2 cone opsin genes and a highly expressed single rod (rh1) opsin gene. Spectral tuning analysis predicted these vitamin A1-based visual photopigments to exhibit spectral absorbance maxima at 461 nm (rh2), 496 nm (rh1) and 555 nm (lws); suggesting the presence of dichromacy in this species. Indeed, P. motoro demonstrates the potential to be equally sensitive to wavelengths from 380 to 600 nm of the visible spectrum. Behavioural results showed that red and green plates, as well as blue and yellow plates, were readily discriminated based on colour; however, brightness differences also played a part in the discrimination of blue and yellow. Red hues of different brightness were distinguished significantly above chance level from one another. In conclusion, the genetic and behavioural results support prior data on marine stingrays. However, this study suggests that freshwater stingrays of the family Potamotrygonidae may have a visual colour system that has ecologically adapted to a riverine habitat.

Serial reversal learning in freshwater stingrays (Potamotrygon motoro).

Serial reversal learning is considered a reliable approach for the testing of behavioral flexibility, and animals that inhabit fluctuating habitats and different environments are expected to possess behavioral and cognitive flexibility. The ocellate river stingray (Potamotrygon motoro) is one such species. Comprising the first serial reversal learning experiment among elasmobranchs, this study trained seven juvenile P. motoro in a visual two-alternative forced-choice task, in which a food-rewarded stimulus and an unrewarded alternative stimulus were presented in pseudo-random order on either side of a barrier. In the session after a stingray reached the learning criterion (LC), food was associated with the alternative stimulus, and this reward association continued to be switched whenever LC was achieved. Overall, five stingrays reversed successfully at least once. All of them required more sessions (mean = 67.8 sessions) for the first reversal than they needed during training (mean = 22 sessions). One stingray demonstrated progressive improvement across four reversal phases, thus showing that Potamotrygon motoro can inhibit a previously learned association faster with experience, probably as it develops strategies for rule identification. One individual became slower to reach the (LC) across three reversal phases, which may indicate a dominant influence of proactive interference. Another stingray demonstrated large fluctuations across three reversals. The fourth individual completed two reversals and required approximately the same number of sessions for each. One stingray had just completed one reversal by the end of this study. Since stingrays took longer than a related species to reverse learning, future studies could look at the effects of changing stimulus type or training regime on the performance of P. motoro.

Something worth remembering: visual discrimination in sharks.

This study investigated memory retention capabilities of juvenile gray bamboo sharks (Chiloscyllium griseum) using two-alternative forced-choice experiments. The sharks had previously been trained in a range of visual discrimination tasks, such as distinguishing between squares, triangles and lines, and their corresponding optical illusions (i.e., the Kanizsa figures or Müller-Lyer illusions), and in the present study, we tested them for memory retention. Despite the absence of reinforcement, sharks remembered the learned information for a period of up to 50 weeks, after which testing was terminated. In fish, as in other vertebrates, memory windows vary in duration depending on species and task; while it may seem beneficial to retain some information for a long time or even indefinitely, other information may be forgotten more easily to retain flexibility and save energy. The results of this study indicate that sharks are capable of long-term memory within the framework of selected cognitive skills. These could aid sharks in activities such as food retrieval, predator avoidance, mate choice or habitat selection and therefore be worth being remembered for extended periods of time. As in other cognitive tasks, intraspecific differences reflected the behavioral breadth of the species.

The shark Chiloscyllium griseum can orient using turn responses before and after partial telencephalon ablation.

This study assessed spatial memory and orientation strategies in Chiloscyllium griseum. In the presence of visual landmarks, six sharks were trained in a fixed turn response. Group 1 started from two possible compartments approaching two goal locations, while group 2 started from and approached only one location, respectively. The learning criterion was reached within 9 ± 5.29 (group 1) and 8.3 ± 3.51 sessions (group 2). Transfer tests revealed that sharks had applied a direction strategy, possibly in combination with some form of place learning. Without visual cues, sharks relied solely on the former. To identify the underlying neural substrate(s), telencephalic were lesioned and performance compared before and after surgery. Ablation of the dorsal and medial pallia only had an effect on one shark (group 1), indicating that the acquisition and retention of previously gained knowledge were unaffected in the remaining four individuals. Nonetheless, the shark re-learned the task. In summary, C. griseum can utilize fixed turn responses to navigate to a goal; there is also some evidence for the use of external visual landmarks while orienting. Probably, strategies can be used alone or in combination. Neither the dorsal nor medial pallium seems to be responsible for the acquisition and processing of egocentric information.

Place learning prior to and after telencephalon ablation in bamboo and coral cat sharks (Chiloscyllium griseum and Atelomycterus marmoratus).

This study assessed complex spatial learning and memory in two species of shark, the grey bamboo shark (Chiloscyllium griseum) and the coral cat shark (Atelomycterus marmoratus). It was hypothesized that sharks can learn and apply an allocentric orientation strategy. Eight out of ten sharks successfully completed the initial training phase (by locating a fixed goal position in a diamond maze from two possible start points) within 14.9 ± 7.6 sessions and proceeded to seven sets of transfer tests, in which sharks had to perform under altered environmental conditions. Transfer tests revealed that sharks had oriented and solved the tasks visually, using all of the provided environmental cues. Unintentional cueing did not occur. Results correspond to earlier studies on spatial memory and cognitive mapping in other vertebrates. Future experiments should investigate whether sharks possess a cognitive spatial mapping system as has already been found in several teleosts and stingrays. Following the completion of transfer tests, sharks were subjected to ablation of most of the pallium, which compromised their previously acquired place learning abilities. These results indicate that the telencephalon plays a crucial role in the processing of information on place learning and allocentric orientation strategies.

Avoidance conditioning in bamboo sharks (Chiloscyllium griseum and C. punctatum): behavioral and neuroanatomical aspects.

Animals face different threats; to survive, they have to anticipate how to react or how to avoid these. It has already been shown in teleosts that selected regions in the telencephalon, i.e., the medial pallium, are involved in avoidance learning strategies. No such study exists for any chondrichthyan. In nature, an avoidance reaction may vary, ranging from a 'freeze' reaction to a startling response and quick escape. This study investigated whether elasmobranchs (Chiloscylliumgriseum and C. punctatum) can be conditioned in an aversive classical conditioning paradigm. Upon successful conditioning, the dorsal, medial and lateral pallium were removed (group 1) and performance tested again. In a second group, the same operation was performed prior to training. While conditioning was successful in individuals of both groups, no escape responses were observed. Post-operative performance was assessed and compared between individual and groups to reveal if the neural substrates governing avoidance behavior or tasks learned in a classical conditioning paradigm are located within the telencephalon, as has been shown for teleosts such as goldfish.

Acoustic discrimination in the grey bamboo shark Chiloscyllium griseum.

Cognitive abilities of sharks are well developed and comparable to teleosts and other vertebrates. Most studies exploring elasmobranch cognitive abilities have used visual stimuli, assessing a wide range of discrimination tasks, memory retention and spatial learning abilities. Some studies using acoustic stimuli in a cognitive context have been conducted, but a basic understanding of sound induced behavioural changes and the underlying mechanisms involved are still lacking. This study explored the acoustic discrimination abilities of seven juvenile grey bamboo sharks (Chiloscyllium griseum) using a Go/No-Go method, which so far had never been tested in sharks before. After this, the smallest frequency difference leading to a change in behaviour in the sharks was studied using a series of transfer tests. Our results show that grey bamboo sharks can learn a Go/No-Go task using both visual and acoustic stimuli. Transfer tests elucidated that, when both stimulus types were presented, both were used. Within the tested range of 90-210 Hz, a frequency difference of 20-30 Hz is sufficient to discriminate the two sounds, which is comparable to results previously collected for sharks and teleosts. Currently, there is still a substantial lack of knowledge concerning the acoustic abilities and sound induced behaviours of sharks while anthropogenic noise is constantly on the rise. New insights into shark sound recognition, detection and use are therefore of the utmost importance and will aid in management and conservation efforts of sharks.

Same or different? Abstract relational concept use in juvenile bamboo sharks and Malawi cichlids.

Sorting objects and events into categories and concepts is an important cognitive prerequisite that spares an individual the learning of every object or situation encountered in its daily life. Accordingly, specific items are classified in general groups that allow fast responses to novel situations. The present study assessed whether bamboo sharks Chiloscyllium griseum and Malawi cichlids Pseudotropheus zebra can distinguish sets of stimuli (each stimulus consisting of two abstract, geometric objects) that meet two conceptual preconditions, i.e., (1) "sameness" versus "difference" and (2) a certain spatial arrangement of both objects. In two alternative forced choice experiments, individuals were first trained to choose two different, vertically arranged objects from two different but horizontally arranged ones. Pair discriminations were followed by extensive transfer test experiments. Transfer tests using stimuli consisting of (a) black and gray circles and (b) squares with novel geometric patterns provided conflicting information with respect to the learnt rule "choose two different, vertically arranged objects", thereby investigating (1) the individuals' ability to transfer previously gained knowledge to novel stimuli and (2) the abstract relational concept(s) or rule(s) applied to categorize these novel objects. Present results suggest that the level of processing and usage of both abstract concepts differed considerably between bamboo sharks and Malawi cichlids. Bamboo sharks seemed to combine both concepts-although not with equal but hierarchical prominence-pointing to advanced cognitive capabilities. Conversely, Malawi cichlids had difficulties in discriminating between symbols and failed to apply the acquired training knowledge on new sets of geometric and, in particular, gray-level transfer stimuli.

Counting on Numbers-Numerical Abilities in Grey Bamboo Sharks and Ocellate River Stingrays.

Over the last decade, studies examining the cognitive abilities of fish have increased, using a broad range of approaches. One of the foci has been to test the ability of fish to discriminate quantities of items and to determine whether fish can solve tasks solely on the basis of numerical information. This study is the first to investigate this ability in two elasmobranch species. All animals were trained in two-alternative forced-choice visual experiments and then examined in transfer tests, to determine if previously gained knowledge could be applied to new tasks. Results show that the grey bamboo shark (Chiloscyllium griseum) and the ocellate river stingray (Potamotrygon motoro) can discriminate quantities based on numerical information alone, while continuous variables were controlled for. Furthermore, the data indicates that similar magnitudes and limits for quantity discrimination exist as in other animals. However, the high degree of intraspecific variation that was observed as well as the low rate of animals proving to be successful suggest that the ability to discriminate quantities may not be as important to these species as to some other vertebrate and invertebrate species tested so far.

Immediate early gene expression related to learning and retention of a visual discrimination task in bamboo sharks (Chiloscyllium griseum).

Using the expression of the immediate early gene (IEG) egr-1 as a neuronal activity marker, brain regions potentially involved in learning and long-term memory functions in the grey bamboo shark were assessed with respect to selected visual discrimination abilities. Immunocytochemistry revealed a significant up-regulation of egr-1 expression levels in a small region of the telencephalon of all trained sharks (i.e., 'early' and 'late learners', 'recallers') when compared to three control groups (i.e., 'controls', 'undisturbed swimmers', 'constant movers'). There was also a well-defined difference in egr-1 expression patterns between the three control groups. Additionally, some staining was observed in diencephalic and mesencephalic sections; however, staining here was weak and occurred only irregularly within and between groups. Therefore, it could have either resulted from unintentional cognitive or non-cognitive inducements (i.e., relating to the mental processes of perception, learning, memory, and judgment, as contrasted with emotional and volitional processes) rather than being a training effect. Present findings emphasize a relationship between the training conditions and the corresponding egr-1 expression levels found in the telencephalon of Chiloscyllium griseum. Results suggest important similarities in the neuronal plasticity and activity-dependent IEG expression of the elasmobranch brain with other vertebrate groups. The presence of the egr-1 gene seems to be evolutionarily conserved and may therefore be particularly useful for identifying functional neural responses within this group.

The Ebbinghaus illusion in the gray bamboo shark (Chiloscyllium griseum) in comparison to the teleost damselfish (Chromis chromis).

This is the first study to comparatively assess the perception of the Ebbinghaus-Titchener circles and variations of the Delboeuf illusion in four juvenile bamboo sharks (Chiloscyllium griseum) and five damselfish (Chromis chromis) using identical training paradigms. We aimed to investigate whether these two species show similarities in the perceptual integration of local elements into the global context. The Ebbinghaus-Titchener circles consist of two equally sized central test circles surrounded by smaller or larger circles of different size, number and/or distance. During training, sharks and damselfish learned to distinguish a large circle from a small circle, regardless (i) of its gray level and (ii) of the presence of surrounding circles arranged along an outer semi-circle. During the subsequent transfer period, individuals were presented with variations of the Ebbinghaus-Titchener circles and the Delboeuf illusion. Similar to adult humans, dolphins, or some birds, damselfish tended to judge the test circle surrounded by smaller inducers as larger than the one surrounded by larger inducers (contrast effect). However, sharks significantly preferred the overall larger figure or chose indifferently between both alternatives (assimilation effect). These contrasting responses point towards potential differences in perceptual processing mechanisms, such as 'filling-in' or '(a)modal completion', 'perceptual grouping', and 'local' or 'global' visual perception. The present study provides intriguing insights into the perceptual abilities of phylogenetically distant taxa separated in evolutionary time by 200 million years.

The brain creates illusions not just for us: sharks (Chiloscyllium griseum) can "see the magic" as well.

Bamboo sharks (Chiloscyllium griseum) were tested for their ability to perceive subjective and illusionary contours as well as line length illusions. Individuals were first trained to differentiate between squares, triangles, and rhomboids in a series of two alternative forced-choice experiments. Transfer tests then elucidated whether Kanizsa squares and triangles, grating gaps and phase shifted abutting gratings were also perceived and distinguished. The visual systems of most vertebrates and even invertebrates perceive illusionary contours despite the absence of physical luminance, color or textural differences. Sharks are no exception to the rule; all tasks were successfully mastered within 3-24 training sessions, with sharks discriminating between various sets of Kanizsa figures and alternative stimuli, as well as between subjective contours in >75% of all tests. However, in contrast to Kanizsa figures and subjective contours, sharks were not deceived by Müller-Lyer (ML) illusions. Here, two center lines of equal length are comparatively set between two arrowheads or -tails, in which case the line featuring the two arrow tails appears to be longer to most humans, primates and birds. In preparation for this experiment, lines of varying length, and lines of unequal length randomly featuring either two arrowheads or -tails on their ends, were presented first. Both sets of lines were successfully distinguished by most sharks. However, during presentation of the ML illusions sharks failed to succeed and succumbed either to side preferences or chose according to chance.

Visual discrimination abilities in the gray bamboo shark (Chiloscyllium griseum).

This study assessed visual discrimination abilities in bamboo sharks (Chiloscyllium griseum). In a visual discrimination task using two-dimensional (2D) geometric stimuli, sharks learned to distinguish between a square, being the positive (rewarded) stimulus, and several negative stimuli, such as two differently sized triangles, a circle, a rhomboid and a cross. Although the amount of sessions to reach the learning criterion and the average trial time needed to solve each new task did not vary significantly, the number of correct choices per session increased significantly with on-going experiments. The results indicate that the sharks did not simply remember the positive stimulus throughout the different training phases. Instead, individuals also seemed to learn each negative symbol and possibly had to "relearn" at least some aspects of the positive stimulus during each training phase. The sharks were able to distinguish between the 2D stimulus pairs at a learning rate corresponding to that found in teleosts. As expected, it took the sharks longer to learn a reversal task (with the positive stimulus now being the negative one) than to discriminate between the other stimulus pairs. Nevertheless, the present results suggest that bamboo sharks can learn visual discrimination tasks, succeed in a reversal task and probably retain (some) information about a previously learned task when progressing to a new one.

Cognitive abilities in Malawi cichlids (Pseudotropheus sp.): matching-to-sample and image/mirror-image discriminations.

The ability to recognize and distinguish between visual stimuli is fundamental for everyday survival of many species. While diverse aspects of cognition, including complex visual discrimination tasks were previously successfully assessed in fish, it remains unknown if fish can learn a matching-to-sample concept using geometrical shapes and discriminate between images and their mirror-image counterparts. For this purpose a total of nine Malawi cichlids (Pseudotropheus sp.) were trained in two matching-to-sample (MTS) and three two-choice discrimination tasks using geometrical, two-dimensional visual stimuli. Two out of the three discrimination experiments focused on the ability to discriminate between images and their mirror-images, the last was a general discrimination test. All fish showed quick associative learning but were unable to perform successfully in a simultaneous MTS procedure within a period of 40 sessions. Three out of eight fish learned to distinguish between an image and its mirror-image when reflected vertically; however none of the fish mastered the task when the stimulus was reflected horizontally. These results suggest a better discrimination ability of vertical compared to horizontal mirror-images, an observation that is widespread in literature on mirror-image discrimination in animals. All fish performed well in the general visual discrimination task, thereby supporting previous results obtained for this species.

Spatial learning and memory retention in the grey bamboo shark (Chiloscyllium griseum).

Like other vertebrates, fish can learn to solve a wide variety of tasks; however, many of their cognitive abilities, particularly in cartilaginous fishes, still remain unknown. This study investigated memory retention capabilities of spatial learning tasks in the grey bamboo shark (Chiloscyllium griseum, n=8) after behavioural training in a two-choice experiment (T-maze). Sharks learned to obtain food at a goal location, either by performing a specific turn response or by using a variety of external landmarks for guidance. Following successful training, sharks were subjected to several training breaks ranging from one to six weeks, after which they were tested again for memory retention capabilities. Sharks successfully mastered the spatial tasks after 5-21 sessions and retained this knowledge in the absence of reinforcement for a period of up to six weeks. This is the first study to show that sharks, like stingrays and other vertebrates, can solve spatial tasks and retain spatial knowledge for an extended period of time, possibly aiding them in activities such as food retrieval, predator avoidance, mate choice and habitat selection.

The role of olfaction throughout juvenile development: functional adaptations in elasmobranchs.

Seven elasmobranch species, a group known for their highly-developed sense of smell, were examined for developmental changes in the number of olfactory lamellae, the size of the surface area of the sensory olfactory epithelium and the mass of both the olfactory rosettes (primary input to the CNS), and the olfactory bulbs. Within each species, juveniles possessed miniature versions of the adult olfactory organs, visually not distinguishable from these and without any obvious structural differences (e.g., with respect to the number of lamellae and the extent of secondary folding) between differently sized individuals. The size of the olfactory organs was positively correlated with body length and body mass, although few species showed proportional size scaling. In Aetobatus narinari and Aptychotrema rostrata, olfactory structures increased in proportion to body size. With respect to the growth of the olfactory bulb, all species showed allometric but not proportional growth. Olfaction may be of particular importance to juveniles in general, which are often subjected to heavy predation rates and fierce inter/intraspecific competition. Accordingly, it would be advantageous to possess a fully functional olfactory system early on in development. Slow growth rates of olfactory structures could then be attributed to a greater reliance on other sensory systems with increasing age or simply be regarded as maintaining an already optimized olfactory system.

Morphometric and ultrastructural comparison of the olfactory system in elasmobranchs: the significance of structure-function relationships based on phylogeny and ecology.

This study investigated the relationship between olfactory morphology, habitat occupancy, and lifestyle in 21 elasmobranch species in a phylogenetic context. Four measures of olfactory capability, that is, the number of olfactory lamellae, the surface area of the olfactory epithelium, the mass of the olfactory bulb, and the mass of the olfactory rosette were compared between individual species and groups, comprised of species with similar habitat and/or lifestyle. Statistical analyses using generalized least squares phylogenetic regression revealed that bentho-pelagic sharks and rays possess significantly more olfactory lamellae and larger sensory epithelial surface areas than benthic species. There was no significant correlation between either olfactory bulb or rosette mass and habitat type. There was also no significant difference between the number of lamellae or the size of the sensory surface area in groups comprised of species with similar diets, that is, groups preying predominantly on crustaceans, cephalopods, echinoderms, polychaetes, molluscs, or teleosts. However, some groups had significantly larger olfactory bulb or rosette masses than others. There was little evidence to support a correlation between phylogeny and morphology, indicating that differences in olfactory capabilities are the result of functional rather than phylogenetic adaptations. All olfactory epithelia exhibited microvilli and cilia, with microvilli in both nonsensory and sensory areas, and cilia only in sensory areas. Cilia over the sensory epithelia originated from supporting cells. In contrast to teleosts, which possess ciliated and microvillous olfactory receptor types, no ciliated olfactory receptor cells were observed. This is the first comprehensive study comparing olfactory morphology to several aspects of elasmobranch ecology in a phylogenetic context.