Characterizing ontogeny of quantity discrimination in zebrafish.

A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here, we investigated the ontogeny of this ability using a group size preference (GSP) task in juvenile zebrafish. Fish showed GSP from 21 days post-fertilization and reliably chose the larger group when presented with discriminations of between 1 versus 3, 2 versus 5 and 2 versus 3 conspecifics but not 2 versus 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1 : 2, fish could discriminate between 1 versus 2 individuals and 3 versus 6, but again, not when given a choice between 2 versus 4 individuals. These findings are in agreement with studies in other species, suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fishes may be the result of an interplay between attentional, cognitive and memory-related mechanisms as in humans and other animals. Our results emphasize the potential of the use of zebrafish to explore the genetic and neural processes underlying the ontogeny and function of number cognition.

Moderate early life stress improves adult zebrafish (Danio rerio) working memory but does not affect social and anxiety-like responses.

Early life stress (ELS) is defined as a short or chronic period of trauma, environmental or social deprivation, which can affect different neurochemical and behavioral patterns during adulthood. Zebrafish (Danio rerio) have been widely used as a model system to understand human neurodevelopmental disorders and display translationally relevant behavioral and stress-regulating systems. In this study, we aimed to investigate the effects of moderate ELS by exposing young animals (6-weeks postfertilization), for 3 consecutive days, to three stressors, and analyzing the impact of this on adult zebrafish behavior (16-week postfertilization). The ELS impact in adults was assessed through analysis of performance on tests of unconditioned memory (free movement pattern Y-maze test), exploratory and anxiety-related task (novel tank diving test), and social cohesion (shoaling test). Here, we show for the first time that moderate ELS increases the number of alternations in turn-direction compared to repetitions in the unconditioned Y-maze task, suggesting increased working memory, but has no effect on shoal cohesion, locomotor profile, or anxiety-like behavior. Overall, our data suggest that moderate ELS may be linked to adaptive flexibility which contributes to build "resilience" in adult zebrafish by improving working memory performance.

Are cerebral and behavioural lateralization related to anxiety-like traits in the animal model zebrafish (Danio rerio)?

Brain lateralization refers to hemispheric asymmetries in functions and/or neuroanatomical structures. Functional specialization in non-human animals has been mainly inferred through observation of lateralized motor responses and sensory perception. Only in a few cases has the influence of brain asymmetries on behaviour been described. Zebrafish has rapidly become a valuable model to investigate this issue as it displays epithalamic asymmetries that have been correlated to some lateralized behaviours. Here we investigated the relation between neuroanatomical or behavioural lateralization and anxiety using a light-dark preference test in adult zebrafish. In Experiment 1, we observed how scototaxis response varied as a function of behavioural lateralization measured in the detour task as turning preference in front of a dummy predator. In Experiment 2, foxD3:GFP transgenic adult zebrafish with left or right parapineal position, were tested in the same light-dark test as fish in Experiment 1. No correlation was found between the behaviour observed in the detour test and in the scototaxis test nor between the left- and right-parapineal fish and the scototaxis response. The consistency of results obtained in both experiments indicates that neither behavioural nor neuroanatomical asymmetries are related to anxiety-related behaviours measured in the light-dark test.

The ontogeny of continuous quantity discrimination in zebrafish larvae (Danio rerio).

Several studies have investigated the ontogeny of the capacity to discriminate between discrete numerical information in human and non-human animals. Contrarily, less attention has been devoted to the development of the capacity to discriminate continuous quantities. Recently, we set up a fast procedure for screening continuous quantity abilities in adult individuals of an animal model in neurodevelopmental research, the zebrafish. Two different sized holes are presented in a wall that divides the home tank in two halves and the spontaneous preference of fish for passing through the larger hole is exploited to measure their discrimination ability. We tested zebrafish larvae in the first, second and third week of life varying the relative size of the smaller circle (0.60, 0.75, 0.86, 0.91 area ratio). We found that the number of passages increased across the age. The capacity to discriminate the larger hole decreased as the ratio between the areas increased. No difference in accuracy was found as a function of age. The accuracy of larval zebrafish almost overlaps that found in adults in a previous study, suggesting a limited role of maturation and experience on the ability to estimate areas in this species.

Food quantity discrimination in puppies (Canis lupus familiaris).

There is considerable evidence that animals are able to discriminate between quantities. Despite the fact that quantitative skills have been extensively studied in adult individuals, research on their development in early life is restricted to a limited number of species. We, therefore, investigated whether 2-month-old puppies could spontaneously discriminate between different quantities of food items. We used a simultaneous two-choice task in which puppies were presented with three numerical combinations of pieces of food (1 vs. 8, 1 vs. 6 and 1 vs. 4), and they were allowed to select only one option. The subjects chose the larger of the two quantities in the 1 vs. 8 and the 1 vs. 6 combinations but not in the 1 vs. 4 combination. Furthermore, the last quantity the puppies looked at before making their choice and the time spent looking at the larger/smaller amounts of food were predictive of the choices they made. Since adult dogs are capable of discriminating between more difficult numerical contrasts when tested with similar tasks, our findings suggest that the capacity to discriminate between quantities is already present at an early age, but that it is limited to very easy discriminations.

Everything is subjective under water surface, too: visual illusions in fish.

The study of visual illusions has captured the attention of comparative psychologists since the last century, given the unquestionable advantage of investigating complex perceptual mechanisms with relatively simple visual patterns. To date, the observation of animal behavior in the presence of visual illusions has been largely confined to mammal and bird studies. Recently, there has been increasing interest in investigating fish, too. The attention has been particularly focused on guppies, redtail splitfin and bamboo sharks. Overall, the tested species were shown to experience a human-like perception of different illusory phenomena involving size, number, motion, brightness estimation and illusory contours. However, in some cases, no illusory effects, or evidence for a reverse illusion, were also reported. Here, we review the current state of the art in this field. We conclude that a wider investigation of visual illusions in fish is fundamental to form a broader comprehension of perceptual systems of vertebrates. Furthermore, we believe that this type of investigation could help us to address general important issues in perceptual studies, such as the role of ecology in shaping perceptual systems, the existence of interindividual variability in the visual perception of nonhuman species and the role of cortical activity in the emergence of visual illusions.

Guppies, Poecilia reticulata, perceive a reversed Delboeuf illusion.

Animals are often required to estimate object sizes during several fitness-related activities, such as choosing mates, foraging, and competing for resources. Some species are susceptible to size illusions, i.e. the misperception of the size of an object based on the surrounding context, but other species are not. This interspecific variation might be adaptive, reflecting species-specific selective pressures; according to this hypothesis, it is important to test species in which size discrimination has a notable ecological relevance. We tested susceptibility to a size illusion in the guppy, Poecilia reticulata, a fish species required to accurately estimate sizes during mate choice, foraging, and antipredator behaviours. We focussed on the Delboeuf illusion, in which an object is typically perceived to be larger when surrounded by a smaller object. In experiment 1, we trained guppies to select the larger of two circles to obtain a food reward and then tested them using stimuli arranged in a Delboeuf-like pattern. In experiment 2, we tested guppies in a spontaneous food choice task to determine whether the subjective size perception of food items is affected by the surrounding context. Jointly, our experiments indicated that guppies perceived the Delboeuf illusion, but in a reverse direction relative to humans: guppies estimated as larger the stimulus that human perceived as smaller. Our results indicated susceptibility to size illusions also in a species required to perform accurate size discrimination and support previous evidence of variability in illusion susceptibility across vertebrates.

Honeybees use absolute rather than relative numerosity in number discrimination.

Various vertebrate species use relative numerosity judgements in comparative assessments of quantities for which they use larger/smaller relationships rather than absolute number. The numerical ability of honeybees shares basic properties with that of vertebrates but their use of absolute or relative numerosity has not been explored. We trained free-flying bees to choose variable images containing three dots; one group ('larger') was trained to discriminate 3 from 2, while another group ('smaller') was trained to discriminate 3 from 4. In both cases, numbers were kept constant but stimulus characteristics and position were varied from trial to trial. Bees were then tested with novel stimuli displaying the previously trained numerosity (3) versus a novel numerosity (4 for 'larger' and 2 for 'smaller'). Both groups preferred the three-item stimulus, consistent with absolute numerosity. They also exhibited ratio-dependent discrimination of numbers, a property shared by vertebrates, as performance after 2 versus 3 was better than after 3 versus 4 training. Thus, bees differ from vertebrates in their use of absolute rather than of relative numerosity but they also have some numeric properties in common.

Sex differences in discrimination reversal learning in the guppy.

In several mammalian and avian species, females show a higher performance than males in tasks requiring cognitive flexibility such as the discrimination reversal learning. A recent study showed that female guppies are twice as efficient as males in a reversal learning task involving yellow-red discrimination, suggesting a higher cognitive flexibility in female guppies. However, the possibility exists that the superior performance exhibited by females does not reflect a general sex difference in cognitive abilities, but instead, is confined to colour discrimination tasks. To address this issue, we compared male and female guppies in two different discrimination reversal learning tasks and we performed a meta-analysis of these experiments and the previous one involving colour discrimination. In the first experiment of this study, guppies were tested in a task requiring them to learn to select the correct arm of a T-maze in order to rejoin a group of conspecifics. In experiment 2, guppies were observed in a numerical task requiring them to discriminate between 5 and 10 dots in order to obtain a food reward. Although females outperformed males in one condition of the T-maze, we did not find any clear evidence of females' greater reversal learning performance in either experiment. However, the meta-analysis of the three experiments supported the hypothesis of females' greater reversal learning ability. Our data do not completely exclude the idea that female guppies have a generally higher cognitive flexibility than males; however, they suggest that the size of this sex difference might depend on the task.

Do domestic dogs (Canis lupus familiaris) perceive the Delboeuf illusion?

In the last decade, visual illusions have been repeatedly used as a tool to compare visual perception among species. Several studies have investigated whether non-human primates perceive visual illusions in a human-like fashion, but little attention has been paid to other mammals, and sensitivity to visual illusions has been never investigated in the dog. Here, we studied whether domestic dogs perceive the Delboeuf illusion. In human and non-human primates, this illusion creates a misperception of item size as a function of its surrounding context. To examine this effect in dogs, we adapted the spontaneous preference paradigm recently used with chimpanzees. Subjects were presented with two plates containing food. In control trials, two different amounts of food were presented in two identical plates. In this circumstance, dogs were expected to select the larger amount. In test trials, equal food portion sizes were presented in two plates differing in size: if dogs perceived the illusion as primates do, they were expected to select the amount of food presented in the smaller plate. Dogs significantly discriminated the two alternatives in control trials, whereas their performance did not differ from chance in test trials with the illusory pattern. The fact that dogs do not seem to be susceptible to the Delboeuf illusion suggests a potential discontinuity in the perceptual biases affecting size judgments between primates and dogs.

Quantitative abilities in a reptile (Podarcis sicula).

The ability to identify the largest amount of prey available is fundamental for optimizing foraging behaviour in several species. To date, this cognitive skill has been observed in all vertebrate groups except reptiles. In this study we investigated the spontaneous ability of ruin lizards to select the larger amount of food items. In Experiment 1, lizards proved able to select the larger food item when presented with two alternatives differing in size (0.25, 0.50, 0.67 and 0.75 ratio). In Experiment 2 lizards presented with two groups of food items (1 versus 4, 2 versus 4, 2 versus 3 and 3 versus 4 items) were unable to select the larger group in any contrast. The lack of discrimination in the presence of multiple items represents an exception in numerical cognition studies, raising the question as to whether reptiles' quantitative abilities are different from those of other vertebrate groups.

Relative versus absolute numerical representation in fish: Can guppies represent "fourness"?

In recent years, the use of operant conditioning procedures has shown that species as diverse as chimpanzees, honeybees, and mosquitofish can be trained to discriminate between sets containing different numbers of objects. However, to succeed in this task, subjects can use two different strategies: either select the array containing a specific number of items (an absolute numerosity rule), or select the set containing the larger (or smaller) quantity of items (a relative numerosity rule). In the latter case, subjects need not only be able to judge whether two numerosities are equal or different but also be able to order numerosities. Here, in two experiments, we address whether fish can perform both kinds of judgment by training them with specific numerosities and testing their generalization to new numerosity contrasts. In Experiment 1, subjects were initially trained to select between visual arrays of 6 and 12 shapes, and were then tested with a contrast pairing the previously trained numerosity (either 6 or 12) with a novel numerosity (respectively, 3 or 24). Spontaneously, subjects selected the novel numerosity, in accordance with a relative numerosity rule. The second experiment tested whether guppies can also learn to select one specific number against all others, if appropriately trained. Fish trained to select an array of 4 shapes against several alternatives (4 vs. 1, 4 vs. 2, 4 vs. 8, 4 vs. 10) learned to recognize the number 4 against all alternatives and proved able to generalize their discrimination to novel, more difficult contrasts (4 vs. 3 and 4 vs. 6 items). In summary, although guppies preferentially opt for relative comparisons, they can flexibly learn either relative or absolute decision criteria on numerosity stimuli, depending on the context.

Numerical acuity of fish is improved in the presence of moving targets, but only in the subitizing range.

There is controversy in comparative psychology about whether on the one hand non-symbolic number estimation of small (≤4) and large numbers involves a single mechanism (an approximate number system), or whether on the other hand enumeration of the numbers 1-4 is accomplished by a separate mechanism, an object tracking system. To date, support for the latter hypothesis has come only from the different ratio-dependency of performance seen in the two numerical ranges, a reading that has been criticized on several grounds. In humans, the two-system hypothesis is supported by evidence showing that manipulation of the physical properties of the stimuli (e.g., the motion of the items) has dissimilar effects on small- and large-number discrimination. In this research, we studied this effect on guppies. Initially, fish were trained to simultaneously discriminate two numerical contrasts having the same easy ratio (0.50): one in the small-number (2 vs. 4) range and one in the large-number (6 vs. 12) range. Half of the fish were presented with moving items; the other half were shown the same stimuli without motion. Fish were then subjected to non-reinforced probe trials in the presence of a more difficult ratio (0.75: 3 vs. 4 and 9 vs. 12). Under both static and moving conditions, the fish significantly discriminated 6 versus 12, but not 9 versus 12 items. As regards small numbers, both groups learned to discriminate a 0.50 ratio, but only fish tested with moving stimuli also discriminated 3 and 4 items. This differential effect suggests that fish may possess two separate systems for small- and large-number discrimination.

Ontogeny of the capacity to compare discrete quantities in fish.

Numerical abilities of adult fish equal or even exceed those of many birds and mammals. The ability to estimate shoal size was previously found to be inborn in guppies, although it improves with age and experience. To study the influence of stimulus variables, here 4- to 9-day-old fish were trained to discriminate between groups of two-dimensional geometric figures when they could use both numbers and cues that co-varied with numerosity (e.g., area) or numerical information only. Subjects rapidly learned to discriminate between quantities under the first condition, but failed to discriminate in the number only condition even with very easy discriminations (1 vs. 4 items). Newborns also failed when stimuli differed in area but not in number, suggesting that, unlike adult fish, newborns can solve this quantity task only if they can use multiple cues. Comparison with shoal discrimination experiments indicates that newborns' capacity to use number is specific to social context. Differences in developmental trajectories suggest the presence in fish of multiple quantification mechanisms which are domain-specific and serve to solve a limited set of problems.

Visual discrimination and amodal completion in zebrafish.

While zebrafish represent an important model for the study of the visual system, visual perception in this species is still less investigated than in other teleost fish. In this work, we validated for zebrafish two versions of a visual discrimination learning task, which is based on the motivation to reach food and companions. Using this task, we investigated zebrafish ability to discriminate between two different shape pairs (i.e., disk vs. cross and full vs. amputated disk). Once zebrafish were successfully trained to discriminate a full from an amputated disk, we also tested their ability to visually complete partially occluded objects (amodal completion). After training, animals were presented with two amputated disks. In these test stimuli, another shape was either exactly juxtaposed or only placed close to the missing sectors of the disk. Only the former stimulus should elicit amodal completion. In human observers, this stimulus causes the impression that the other shape is occluding the missing sector of the disk, which is thus perceived as a complete, although partially hidden, disk. In line with our predictions, fish reinforced on the full disk chose the stimulus eliciting amodal completion, while fish reinforced on the amputated disk chose the other stimulus. This represents the first demonstration of amodal completion perception in zebrafish. Moreover, our results also indicated that a specific shape pair (disk vs. cross) might be particularly difficult to discriminate for this species, confirming previous reports obtained with different procedures.

Quantity as a Fish Views It: Behavior and Neurobiology.

An ability to estimate quantities, such as the number of conspecifics or the size of a predator, has been reported in vertebrates. Fish, in particular zebrafish, may be instrumental in advancing the understanding of magnitude cognition. We review here the behavioral studies that have described the ecological relevance of quantity estimation in fish and the current status of the research aimed at investigating the neurobiological bases of these abilities. By combining behavioral methods with molecular genetics and calcium imaging, the involvement of the retina and the optic tectum has been documented for the estimation of continuous quantities in the larval and adult zebrafish brain, and the contributions of the thalamus and the dorsal-central pallium for discrete magnitude estimation in the adult zebrafish brain. Evidence for basic circuitry can now be complemented and extended to research that make use of transgenic lines to deepen our understanding of quantity cognition at genetic and molecular levels.

The Challenge of Illusory Perception of Animals: The Impact of Methodological Variability in Cross-Species Investigation.

Although we live on the same planet, there are countless different ways of seeing the surroundings that reflect the different individual experiences and selective pressures. In recent decades, visual illusions have been used in behavioural research to compare the perception between different vertebrate species. The studies conducted so far have provided contradictory results, suggesting that the underlying perceptual mechanisms may differ across species. Besides the differentiation of the perceptual mechanisms, another explanation could be taken into account. Indeed, the different studies often used different methodologies that could have potentially introduced confounding factors. In fact, the possibility exists that the illusory perception is influenced by the different methodologies and the test design. Almost every study of this research field has been conducted in laboratories adopting two different methodological approaches: a spontaneous choice test or a training procedure. In the spontaneous choice test, a subject is presented with biologically relevant stimuli in an illusory context, whereas, in the training procedure, a subject has to undergo an extensive training during which neutral stimuli are associated with a biologically relevant reward. Here, we review the literature on this topic, highlighting both the relevance and the potential weaknesses of the different methodological approaches.

Learning by Doing: The Use of Distance, Corners and Length in Rewarded Geometric Tasks by Zebrafish (Danio rerio).

Zebrafish spontaneously use distance and directional relationships among three-dimensional extended surfaces to reorient within a rectangular arena. However, they fail to take advantage of either an array of freestanding corners or an array of unequal-length surfaces to search for a no-longer-present goal under a spontaneous cued memory procedure, being unable to use the information supplied by corners and length without some kind of rewarded training. The present study aimed to tease apart the geometric components characterizing a rectangular enclosure under a procedure recruiting the reference memory, thus training zebrafish in fragmented layouts that provided differences in surface distance, corners, and length. Results showed that fish, besides the distance, easily learned to use both corners and length if subjected to a rewarded exit task over time, suggesting that they can represent all the geometrically informative parts of a rectangular arena when consistently exposed to them. Altogether, these findings highlight crucially important issues apropos the employment of different behavioral protocols (spontaneous choice versus training over time) to assess spatial abilities of zebrafish, further paving the way to deepen the role of visual and nonvisual encodings of isolated geometric components in relation to macrostructural boundaries.

Stimulus characteristics, learning bias and visual discrimination in zebrafish (Danio rerio).

Zebrafish is an emerging model in the study of brain function; however, knowledge about its behaviour and cognition is incomplete. Previous studies suggest this species has limited ability in visual learning tasks compared to other teleosts. In this study, we systematically examined zebrafish's ability to learn to discriminate colour, shape, size, and orientation of figures using an appetitive conditioning paradigm. Contrary to earlier reports, the zebrafish successfully completed all tasks. Not all discriminations were learned with the same speed and accuracy. Subjects discriminated the size of objects better than their shape or colour. In all three tasks, they were faster and more accurate when required to discriminate between outlined figures than between filled figures. With stimuli consisting of outlines, the learning performance of zebrafish was comparable to that observed in higher vertebrates. Zebrafish easily learned a horizontal-vertical discrimination task, but like many other vertebrates, they had great difficulty discriminating a figure from its mirror image. Performance was more accurate for subjects reinforced on one stimulus (green over red, triangle over circle, large over small). Unexpectedly, these stimulus biases occurred only when zebrafish were tested with filled figures, suggesting some causal relationship between stimulus preference, learning bias and performance.

Stress reactivity elicits a tissue-specific reduction in telomere length in aging zebrafish (Danio rerio).

Individual differences in personality are associated with variation in healthy aging. Health behaviours are often cited as the likely explanation for this association; however, an underlying biological mechanism may also exist. Accelerated leukocyte telomere shortening is implicated in multiple age-related diseases and is associated with chronic activation of the hypothalamus-pituitary-adrenal (HPA) axis, providing a link between stress-related personality differences and adverse health outcomes. However, the effects of the HPA axis are tissue specific. Thus, leukocyte telomere length may not accurately reflect telomere length in disease-relevant tissues. Here, we examined the correlation between stress reactivity and telomere length in heart and brain tissue in young (6-9 month) and aging (18 month) zebrafish. Stress reactivity was assessed by tank diving and through gene expression. Telomere length was assessed using quantitative PCR. We show that aging zebrafish have shorter telomeres in both heart and brain. Telomere length was inversely related to stress reactivity in heart but not brain of aging individuals. These data support the hypotheses that an anxious predisposition contributes to accelerated telomere shortening in heart tissue, which may have important implications for our understanding of age-related heart disease, and that stress reactivity contributes to age-related telomere shortening in a tissue-specific manner.