Exploring the Importance of Environmental Complexity for Newly Hatched Zebrafish.

The effects of an early impoverished social or physical environment on vertebrate neural development and cognition has been known for decades. While existing studies have focused on the long-term effects, measuring adult cognitive phenotypes, studies on the effects of environmental complexity on the early stages of development are lacking. Zebrafish (Danio rerio) hatchlings are assumed to have minimal interaction with their environment and are routinely reared in small, bare containers. To investigate the effects of being raised under such conditions on development of behaviour and cognition, hatchlings housed for 10 days in either an enriched or a standard environment underwent two cognitive tasks. The results were mixed. Subjects of the two treatments did not differ in performance when required to discriminate two areas. Conversely, we found a significant effect in a number discrimination task, with subjects from impoverished condition performing significantly worse. In both experiments, larvae reared in impoverished environment showed a reduced locomotor activity. Given the effects that enrichment appears to exert on larvae, a third experiment explored whether hatchlings exhibit a spontaneous preference for more complex environments. When offered a choice between a bare setting and one with objects of different shapes and colors, larvae spent over 70% of time in the enriched sector. Deepening these effects of an early impoverished environment on cognitive development is crucial for the welfare of captive zebrafish populations and for enhancing the quality and reliability of studies on larval zebrafish.

Shortest path choice in zebrafish (Danio rerio).

Many animals regularly move between different locations within their home range. During these journeys, individuals are expected to use the shortest path, because this strategy minimizes energy expenditure and reduces exposure to adverse conditions, such as predation. The ability to find the shortest distance route has been demonstrated in ants, migrating birds and a few mammals. We investigated whether a freshwater fish, Danio rerio, exhibits this ability. Small groups of zebrafish were allowed to move between the two compartments of their tank using two paths differing in length. They developed a preference for the shorter path gradually over the six days of the experiment. Subjects' accuracy in choosing the shorter path varied from below 60%, with a 20% length disparity, to 80% when one path was twice as long as the other. In a second experiment, zebrafish were initially allowed to practice in groups and then tested individually. We found evidence of individual and sex differences in performance, with males performing more accurately than females. However, due to our experimental design, we cannot conclusively determine whether these differences are indeed cognitive or influenced by confounding factors during the group phase of the experiment.

Interspecific differences in developmental mode determine early cognitive abilities in teleost fish.

Most studies on developmental variation in cognition have suggested that individuals are born with reduced or absent cognitive abilities, and thereafter, cognitive performance increases with age during early development. However, these studies have been mainly performed in altricial species, such as humans, in which offspring are extremely immature at birth. In this work, we tested the hypothesis that species with other developmental modes might show different patterns of cognitive development. To this end, we analysed inhibitory control performance in two teleost species with different developmental modes, the zebrafish Danio rerio and the guppy Poecilia reticulata, exploiting a simple paradigm based on spontaneous behaviour and therefore applicable to subjects of different ages. Zebrafish hatch as larvae 3 days after fertilisation, and have an immature nervous system, a situation that mirrors extreme altriciality. We found that at the early stages of development, zebrafish displayed no evidence of inhibitory control, which only begun to emerge after one month of life. Conversely, guppies, which are born after approximately one month of gestation as fully developed and independent individuals, solved the inhibitory control task since their first days of life, although performance increased with sexual maturation. Our study suggests that the typical progression described during early ontogeny in humans and other species might not be the only developmental trend for animals' cognition and that a species' developmental mode might determine variation in cognition across subjects of different age.

Quantity discrimination in newly hatched zebrafish suggests hardwired numerical abilities.

An intriguing hypothesis to explain the ubiquity of numerical abilities is that all vertebrates are born with hardwired neuronal networks for processing numbers. To date, only studies on human foetuses have clearly supported this hypothesis. Zebrafish hatch 48-72 h after fertilisation with an embryonic nervous system, providing a unique opportunity for investigating this hypothesis. Here, we demonstrated that zebrafish larvae exposed to vertical bars at birth acquired an attraction for bar stimuli and we developed a numerical discrimination task based on this preference. When tested with a series of discriminations of increasing difficulty (1vs.4, 1vs.3, 1vs.2, and 2vs.4 bars), zebrafish larvae reliably selected the greater numerosity. The preference was significant when stimuli were matched for surface area, luminance, density, and convex hull, thereby suggesting a true capacity to process numerical information. Converging results from two phylogenetically distant species suggests that numerical abilities might be a hallmark feature of vertebrates' brains.

Extra food provisioning does not affect behavioral lateralization in nestling lesser kestrels.

Costs and benefits of brain lateralization may depend on environmental conditions. Growing evidence indicates that the development of brain functional asymmetries is adaptively shaped by the environmental conditions experienced during early life. Food availability early in life could act as a proxy of the environmental conditions encountered during adulthood, but its potential modulatory effect on lateralization has received little attention. We increased food supply from egg laying to early nestling rearing in a wild population of lesser kestrels Falco naumanni, a sexually dimorphic raptor, and quantified the lateralization of preening behavior (head turning direction). As more lateralized individuals may perform better in highly competitive contexts, we expected that extra food provisioning, by reducing the level of intra-brood competition for food, would reduce the strength of lateralization. We found that extra food provisioning improved nestling growth, but it did not significantly affect the strength or direction of nestling lateralization. In addition, maternal body condition did not explain variation in nestling lateralization. Independently of extra food provisioning, the direction of lateralization differed between the sexes, with female nestlings turning more often toward their right. Our findings indicate that early food availability does not modulate behavioral lateralization in a motor task, suggesting limited phenotypic plasticity in this trait.

Learning and visual discrimination in newly hatched zebrafish.

With the exception of humans, early cognitive development has been thoroughly investigated only in precocial species, well developed at birth and with a broad behavioral and cognitive repertoire. We investigated another highly altricial species, the zebrafish, Danio rerio, whose embryonic development is very rapid (< 72 h). The hatchlings' nervous system is poorly developed, and their cognitive capacities are largely unknown. Larvae trained at 8 days post fertilization rapidly learned to associate a visual pattern with a food reward, showing significant performance at 10 days post fertilization. We exploited this ability to study hatchlings' discrimination learning capacities. Larvae rapidly and accurately learned color and shape discriminations. They also discriminated a figure from its mirror image and from its 90°-rotated version, although with lower performance. Our study revealed impressive similarities in learning and visual discrimination capacities between newborn and adult zebrafish, despite their enormous differences in brain size and degree of development.

Environmental enrichment decreases anxiety-like behavior in zebrafish larvae.

The development of anxiety disorders is often linked to individuals' negative experience. In many animals, development of anxiety-like behavior is modeled by manipulating individuals' exposure to environmental enrichment. We investigated whether environmental enrichment during early ontogenesis affects anxiety-like behavior in larval zebrafish. Larvae were exposed from hatching to either an environment enriched with 3D-objects of different color and shape or to a barren environment. Behavioral testing was conducted at different intervals during development (7, 14, and 21 days post-fertilization, dpf). In a novel object exploration test, 7 dpf larvae of the two treatments displayed similar avoidance of the visual stimulus. However, at 14 and 21 dpf, larvae of the enriched environment showed less avoidance, indicating lower anxiety response. Likewise, larvae of the two treatments demonstrated comparable avoidance of a novel odor stimulus at 7 dpf, with a progressive reduction of anxiety behavior in the enriched treatment with development. In a control experiment, larvae treated before 7 dpf but tested at 14 dpf showed the effect of enrichment on anxiety, suggesting an early determination of the anxiety phenotype. This study confirms a general alteration of zebrafish anxiety-like behavior due to a short enrichment period in first days of life.

Zebrafish excel in number discrimination under an operant conditioning paradigm.

Numerical discrimination is widespread in vertebrates, but this capacity varies enormously between the different species examined. The guppy (Poecilia reticulata), the only teleost examined following procedures that allow a comparison with the other vertebrates, outperforms amphibians, reptiles and many warm-blooded vertebrates, but it is unclear whether this is a feature shared with the other teleosts or represents a peculiarity of this species. We trained zebrafish (Danio rerio) to discriminate between numbers differing by one unit, varying task difficulty from 2 versus 3 to 5 versus 6 items. Non-numerical variables that covary with number, such as density or area, did not affect performance. Most fish reached learning criterion on all tasks up to 4 versus 5 discrimination with no sex difference in accuracy. Although no individual reached learning criterion in the 5 versus 6 task, performance was significant at the group level, suggesting that this may represent the discrimination threshold for zebrafish. Numerosity discrimination abilities of zebrafish compare to those of guppy, being higher than in some warm-blooded vertebrates, such as dogs, horses and domestic fowl, though lower than in parrots, corvids and primates. Learning rate was similar in a control group trained to discriminate between different-sized shapes, but zebrafish were slightly more accurate when discriminating areas than numbers and males were more accurate than females. At the end of the experiment, fish trained on numbers and controls trained on areas generalized to the reciprocal set of stimuli, indicating they had used a relational strategy to solve these tasks.

Guppies (Poecilia reticulata) are deceived by visual illusions during obstacle negotiation.

Animals travelling in their natural environment repeatedly encounter obstacles that they can either detour or go through. Gap negotiation requires an accurate estimation of the opening's size to avoid getting stuck or being injured. Research on visual illusions has revealed that in some circumstances, transformation rules used to generate a three-dimensional representation from bidimensional retinal images fail, leading to systematic errors in perception. In Ebbinghaus and Delboeuf illusions, the presence of task-irrelevant elements causes us to misjudge an object's size. Susceptibility to these illusions was observed in other animals, although with large intraspecific differences. In this study, we investigated whether fish can accurately estimate gap size and whether during this process they may be deceived by illusory patterns. Guppies were extremely accurate in gap negotiation, discriminating holes with a 10% diameter difference. When presented with two identical holes surrounded by inducers to produce Ebbinghaus and Delboeuf patterns, guppies misperceived gap size in the predicted direction. So far, researchers have principally considered illusions as useful tools for exploring the cognitive processing underlying vision. Our findings highlight the possibility that they have important ecological implications, affecting the everyday interactions of an animal with its physical world besides its intra- and interspecific relationships.

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.

Automated Operant Conditioning Devices for Fish. Do They Work?

The growing use of teleosts in comparative cognition and in neurobiological research has prompted many researchers to develop automated conditioning devices for fish. These techniques can make research less expensive and fully comparable with research on warm-blooded species, in which automated devices have been used for more than a century. Tested with a recently developed automated device, guppies (Poecilia reticulata) easily performed 80 reinforced trials per session, exceeding 80% accuracy in color or shape discrimination tasks after only 3-4 training session, though they exhibit unexpectedly poor performance in numerical discrimination tasks. As several pieces of evidence indicate, guppies possess excellent numerical abilities. In the first part of this study, we benchmarked the automated training device with a standard manual training procedure by administering the same set of tasks, which consisted of numerical discriminations of increasing difficulty. All manually-trained guppies quickly learned the easiest discriminations and a substantial percentage learned the more difficult ones, such as 4 vs. 5 items. No fish trained with the automated conditioning device reached the learning criterion for even the easiest discriminations. In the second part of the study, we introduced a series of modifications to the conditioning chamber and to the procedure in an attempt to improve its efficiency. Increasing the decision time, inter-trial interval, or visibility of the stimuli did not produce an appreciable improvement. Reducing the cognitive load of the task by training subjects first to use the device with shape and color discriminations, significantly improved their numerical performance. Allowing the subjects to reside in the test chamber, which likely reduced the amount of attentional resources subtracted to task execution, also led to an improvement, although in no case did subjects match the performance of fish trained with the standard procedure. Our results highlight limitations in the capacity of small laboratory teleosts to cope with operant conditioning automation that was not observed in laboratory mammals and birds and that currently prevent an easy and straightforward comparison with other vertebrates.

Continuous versus discrete quantity discrimination in dune snail (Mollusca: Gastropoda) seeking thermal refuges.

The ability of invertebrates to discriminate quantities is poorly studied, and it is unknown whether other phyla possess the same richness and sophistication of quantification mechanisms observed in vertebrates. The dune snail, Theba pisana, occupies a harsh habitat characterised by sparse vegetation and diurnal soil temperatures well above the thermal tolerance of this species. To survive, a snail must locate and climb one of the rare tall herbs each dawn and spend the daytime hours in an elevated refuge position. Based on their ecology, we predicted that dune snails would prefer larger to smaller groups of refuges. We simulated shelter choice under controlled laboratory conditions. Snails' acuity in discriminating quantity of shelters was comparable to that of mammals and birds, reaching the 4 versus 5 item discrimination, suggesting that natural selection could drive the evolution of advanced cognitive abilities even in small-brained animals if these functions have a high survival value. In a subsequent series of experiments, we investigated whether snails used numerical information or based their decisions upon continuous quantities, such as cumulative surface, density or convex hull, which co-varies with number. Though our results tend to underplay the role of these continuous cues, behavioural data alone are insufficient to determine if dune snails were using numerical information, leaving open the question of whether gastropod molluscans possess elementary abilities for numerical processing.

Poor numerical performance of guppies tested in a Skinner box.

We tested the hypothesis that part of the gap in numerical competence between fish and warm-blooded vertebrates might be related to the more efficient procedures (e.g. automated conditioning chambers) used to investigate the former and could be filled by adopting an adapted version of the Skinner box in fish. We trained guppies in a visual numerosity discrimination task, featuring two difficulty levels (3 vs. 5 and 3 vs. 4) and three conditions of congruency between numerical and non-numerical cues. Unexpectedly, guppies trained with the automated device showed a much worse performance compared to previous investigations employing more "ecological" procedures. Statistical analysis indicated that the guppies overall chose the correct stimulus more often than chance; however, their average accuracy did not exceed 60% correct responses. Learning measured as performance improvement over training was significant only for the stimuli with larger numerical difference. Additionally, the target numerosity was selected more often than chance level only for the set of stimuli in which area and number were fully congruent. Re-analysis of prior studies indicate that the gap between training with the Skinner box and with a naturalistic setting was present only for numerical discriminations, but not for colour and shape discriminations. We suggest that applying automated conditioning chambers to fish might increase cognitive load and therefore interfere with achievement of numerosity discriminations.

Susceptibility to Size Visual Illusions in a Non-Primate Mammal (Equus caballus).

The perception of different size illusions is believed to be determined by size-scaling mechanisms that lead individuals to extrapolate inappropriate 3D information from 2D stimuli. The Muller-Lyer illusion represents one of the most investigated size illusions. Studies on non-human primates showed a human-like perception of this illusory pattern. To date, it is not clear whether non-primate mammals experience a similar illusory effect. Here, we investigated whether horses perceive the Muller-Lyer illusion by using their spontaneous preference for the larger portion of carrot. In control trials, we presented horses with two carrot sticks of different sizes, and in test trials, carrot sticks of identical size were shown to the subjects together with arrowheads made of plastic material and arranged in a way meant to elicit the Müller-Lyer illusion in human observers. In control trials, horses significantly discriminated between the smaller and larger carrot stick. When presented with the illusion, they showed a significant preference for the carrot that humans perceive as longer. Further control trials excluded the possibility that their choices were based on the total size of the carrot stick and the arrowheads together. The susceptibility of horses to this illusion indicates that the perceptual mechanisms underlying size estimation in perissodactyla might be similar to those of primates, notwithstanding the considerable evolutionary divergence in the visual systems of these two mammalian groups.

The devil is in the detail: Zebrafish learn to discriminate visual stimuli only if salient.

Due to their unique characteristics, the zebrafish plays a key role in the comprehension of neurobiology of cognition and its pathologies, such as neurodegenerative diseases. More and more molecular tools for this aim are being developed, but our knowledge about the cognitive abilities of zebrafish remains extremely scarce compared to other teleost fish. We aimed to investigate the complex cognitive abilities of zebrafish using a tracking-based automated conditioning chamber that allowed precise experimental control, avoided potential cueing provided by the observer (Clever Hans phenomenon), and was shown to considerably improve learning in other teleosts. A computer presented two visual stimuli in two sectors of the chamber, and zebrafish had to enter the correct sector to obtain a food reward. Zebrafish quickly learned to use the conditioning device and easily performed up to 80 trials per day. In Experiment 1, zebrafish efficiently discriminated between two differently coloured sides, reaching a 75 % accuracy in only 10 training sessions. Surprisingly, zebrafish failed to choose the correct chamber when the stimuli were two shapes, a small circle and a small triangle, even when, in Experiment 2, training on shape discrimination was prolonged for up to 30 sessions. In Experiment 3, we tested the hypothesis that simultaneously learning to use the conditioning chamber and learning discrimination imposes a too-high cognitive load. However, zebrafish that first successfully learned how the conditioning chamber functioned (in the colour discrimination) subsequently failed in the shape discrimination. Conversely, zebrafish that firstly failed the shape discrimination subsequently learned colour discrimination. In Experiment 4, zebrafish showed some evidence of learning when the stimuli were two large shapes, suggesting that zebrafish did not discriminate between the shapes of the previous experiments because they were not salient enough. Altogether, results suggest constraints in the discrimination learning abilities of zebrafish, which should be taken into account when developing cognitive tasks for this species.

Vegetation cover induces developmental plasticity of lateralization in tadpoles.

Lateralization of cognitive functions influences a large number of fitness-related behaviors and shows, in most species, substantial variation in strength and direction. Laboratory works and field data have suggested that this variation is often due to adaptive phenotypic plasticity. Strong lateralization should be favored in some ecological conditions, for example, under high risk of predation. For anuran tadpoles, the presence of cover affects predation risk, with tadpoles being more exposed to predators in environments with reduced cover. We tested the hypothesis that the amount of cover experienced early in life affects lateralization in the edible frog, Pelophylax esculentus, tadpoles. We exposed embryos and larvae to high or low vegetation cover environments. For half of the subjects, the treatment was constant whereas the remaining subjects were switched to the opposite treatment after hatching. In agreement with the theoretical expectation, tadpoles exposed to low vegetation cover for the entire development were more lateralized and showed a stronger alignment in directionality of lateralization compared with tadpoles exposed to high vegetation cover. This indicates a possible role of natural variation in vegetation abundance and developmental plasticity as determinants of between-population and between-individual differences in lateralization. We also found that shifting from high to low vegetation cover treatments and vice versa disrupted lateralization alignment, suggesting that developmental trajectories for this trait are determined at the embryonic stage and need environmental stability to be fully expressed.

Measuring recognition memory in zebrafish larvae: issues and limitations.

Recognition memory is the capacity to recognize previously encountered objects, events or places. This ability is crucial for many fitness-related activities, and it appears very early in the development of several species. In the laboratory, recognition memory is most often investigated using the novel object recognition test (NORt), which exploits the tendency of most vertebrates to explore novel objects over familiar ones. Despite that the use of larval zebrafish is rapidly increasing in research on brain, cognition and neuropathologies, it is unknown whether larvae possess recognition memory and whether the NORt can be used to assess it. Here, we tested a NOR procedure in zebrafish larvae of 7-, 14- and 21-days post-fertilization (dpf) to investigate when recognition memory first appears during ontogeny. Overall, we found that larvae explored a novel stimulus longer than a familiar one. This response was fully significant only for 14-dpf larvae. A control experiment evidenced that larvae become neophobic at 21-dpf, which may explain the poor performance at this age. The preference for the novel stimulus was also affected by the type of stimulus, being significant with tri-dimensional objects varying in shape and bi-dimensional geometrical figures but not with objects differing in colour. Further analyses suggest that lack of effect for objects with different colours was due to spontaneous preference for one colour. This study highlights the presence of recognition memory in zebrafish larvae but also revealed non-cognitive factors that may hinder the application of NORt paradigms in the early developmental stages of zebrafish.

Male and female guppies differ in problem-solving abilities.

In a number of species, males and females have different ecological roles and therefore might be required to solve different problems. Studies on humans have suggested that the 2 sexes often show different efficiencies in problem-solving tasks; similarly, evidence of sex differences has been found in 2 other mammalian species. Here, we assessed whether a teleost fish species, the guppy, Poecilia reticulata, displays sex differences in the ability to solve problems. In Experiment 1, guppies had to learn to dislodge a disc that occluded a feeder from which they had been previously accustomed to feed. In Experiment 2, guppies had to solve a version of the detour task that required them to learn to enter a transparent cylinder from the open sides to reach a food reward previously freely available. We found evidence of sex differences in both problem-solving tasks. In Experiment 1, females clearly outperformed males, and in Experiment 2, guppies showed a reversed but smaller sex difference. This study indicates that sex differences may play an important role in fish's problem-solving similar to what has previously been observed in some mammalian species.

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.

Guppies show sex and individual differences in the ability to inhibit behaviour.

In humans, individual and sex differences have been long reported for several cognitive tasks and are at least in part due to variability in the function that inhibits behaviour (i.e. inhibitory control). Similar evidence of individual and sex differences in inhibitory abilities is also present in other vertebrates, but is scarce outside primates. Experiments on reversal learning, which requires inhibiting behaviours, suggest that this variability may exist in a teleost fish, the guppy, Poecilia reticulata. We tested this hypothesis by observing guppies in an inhibitory task. Guppies were exposed to unreachable prey inside a transparent tube for six trials. Guppies showed a marked reduction in the number of attempts to catch the prey within the first trial and also over repeated trials. We found a striking sex difference in the capacity to inhibit foraging behaviour. Males attempted to attack the prey twice as often as females and showed negligible improvement over trials. Irrespective of sex, individuals remarkably differed in their performance, with some guppies being systematically more skilled than others across the repeated trials. These results confirm that individual and sex differences in the ability to inhibit behaviour are not restricted to humans and other primates, suggesting that they might be widespread among vertebrates. Variability in inhibitory ability provides an explanation for emerging records of variability in other cognitive tasks in fish.