Environmental conditions shape learning in larval zebrafish.

Growing evidence reveals notable phenotypic plasticity in cognition among teleost fishes. One compelling example is the positive impact of enriched environments on learning performance. Most studies on this effect have focused on juvenile or later life stages, potentially overlooking the importance of early life plasticity. To address this gap, we investigated whether cognitive plasticity in response to environmental factors emerges during the larval stage in zebrafish. Our findings indicate that larvae exposed to an enriched environment after hatching exhibited enhanced habituation learning performance compared to their counterparts raised in a barren environment. This work underscores the presence of developmental phenotypic plasticity in cognition among teleost fish, extending its influence to the very earliest stages of an individual's life.

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.

Enhancing Rearing of European Seabass (Dicentrarchus labrax) in Aquaponic Systems: Investigating the Effects of Enriched Black Soldier Fly (Hermetia illucens) Prepupae Meal on Fish Welfare and Quality Traits.

Within the modern aquaculture goals, the present study aimed to couple sustainable aquafeed formulation and culturing systems. Two experimental diets characterized by 3 and 20% of fish meal replacement with full-fat spirulina-enriched black soldier fly (Hermetia illucens) prepupae meal (HPM3 and HPM20, respectively) were tested on European seabass (Dicentrarchus labrax) juveniles during a 90-day feeding trial performed in aquaponic systems. The experimental diets ensured 100% survival and proper zootechnical performance. No behavioral alterations were evidenced in fish. Histological and molecular analyses did not reveal structural alterations and signs of inflammation at the intestinal level, highlighting the beneficial role on gut health of bioactive molecules typical of HPM or derived from the enriching procedure of insects' growth substrate with spirulina. Considering the quality traits, the tested experimental diets did not negatively alter the fillet's fatty acid profile and did not compromise the fillet's physical features. In addition, the results highlighted a possible role of spirulina-enriched HPM in preserving the fillet from lipid oxidation. Taken together, these results corroborate the use of sustainable ingredients (spirulina-enriched HPM) in aquaponic systems for euryhaline fish rearing.

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.

Individual differences and knockout in zebrafish reveal similar cognitive effects of BDNF between teleosts and mammals.

The remarkable similarities in cognitive performance between teleosts and mammals suggest that the underlying cognitive mechanisms might also be similar in these two groups. We tested this hypothesis by assessing the effects of the brain-derived neurotrophic factor (BDNF), which is critical for mammalian cognitive functioning, on fish's cognitive abilities. We found that individual differences in zebrafish's learning abilities were positively correlated with bdnf expression. Moreover, a CRISPR/Cas9 mutant zebrafish line that lacks the BDNF gene (bdnf-/-) showed remarkable learning deficits. Half of the mutants failed a colour discrimination task, whereas the remaining mutants learned the task slowly, taking three times longer than control bdnf+/+ zebrafish. The mutants also took twice as long to acquire a T-maze task compared to control zebrafish and showed difficulties exerting inhibitory control. An analysis of habituation learning revealed that cognitive impairment in mutants emerges early during development, but could be rescued with a synthetic BDNF agonist. Overall, our study indicates that BDNF has a similar activational effect on cognitive performance in zebrafish and in mammals, supporting the idea that its function is conserved in vertebrates.

Illusional Perspective across Humans and Bees.

For two centuries, visual illusions have attracted the attention of neurobiologists and comparative psychologists, given the possibility of investigating the complexity of perceptual mechanisms by using relatively simple patterns. Animal models, such as primates, birds, and fish, have played a crucial role in understanding the physiological circuits involved in the susceptibility of visual illusions. However, the comprehension of such mechanisms is still a matter of debate. Despite their different neural architectures, recent studies have shown that some arthropods, primarily Hymenoptera and Diptera, experience illusions similar to those humans do, suggesting that perceptual mechanisms are evolutionarily conserved among species. Here, we review the current state of illusory perception in bees. First, we introduce bees' visual system and speculate which areas might make them susceptible to illusory scenes. Second, we review the current state of knowledge on misperception in bees (Apidae), focusing on the visual stimuli used in the literature. Finally, we discuss important aspects to be considered before claiming that a species shows higher cognitive ability while equally supporting alternative hypotheses. This growing evidence provides insights into the evolutionary origin of visual mechanisms across species.

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.

Quantitative abilities of invertebrates: a methodological review.

Quantitative abilities are widely recognized to play important roles in several ecological contexts, such as foraging, mate choice, and social interaction. Indeed, such abilities are widespread among vertebrates, in particular mammals, birds, and fish. Recently, there has been an increasing number of studies on the quantitative abilities of invertebrates. In this review, we present the current knowledge in this field, especially focusing on the ecological relevance of the capacity to process quantitative information, the similarities with vertebrates, and the different methods adopted to investigate this cognitive skill. The literature argues, beyond methodological differences, a substantial similarity between the quantitative abilities of invertebrates and those of vertebrates, supporting the idea that similar ecological pressures may determine the emergence of similar cognitive systems even in distantly related species.

Innate visual discrimination abilities of zebrafish larvae.

The ability to discriminate between objects visually plays a key role in animals' interactions with their environment because it enables them to recognise companions, prey, and predators. In the zebrafish, Danio rerio, hatching occurs early on during development (48-72 h post fertilisation), and the larvae must forage and evade predators despite their immature sensory and cognitive systems. Using a preference paradigm, we investigated whether larval zebrafish are nonetheless capable of discriminating between visual stimuli. We found that larvae discriminated not only between figures with different colours or different shapes, but also between two identical figures with different orientations and between sets of figures with different numerosities. By manipulating larvae's exposure to objects before the test, we demonstrated that their discrimination abilities are innate and do not depend upon experience. This study highlighted that zebrafish possess relatively sophisticated visual discrimination abilities even at the larval stage. These abilities likely improve larval survival via the recognition of biologically relevant stimuli.

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.

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.

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.

Bumblebees Use Sequential Scanning of Countable Items in Visual Patterns to Solve Numerosity Tasks.

Most research in comparative cognition focuses on measuring if animals manage certain tasks; fewer studies explore how animals might solve them. We investigated bumblebees' scanning strategies in a numerosity task, distinguishing patterns with two items from four and one from three, and subsequently transferring numerical information to novel numbers, shapes, and colors. Video analyses of flight paths indicate that bees do not determine the number of items by using a rapid assessment of number (as mammals do in "subitizing"); instead, they rely on sequential enumeration even when items are presented simultaneously and in small quantities. This process, equivalent to the motor tagging ("pointing") found for large number tasks in some primates, results in longer scanning times for patterns containing larger numbers of items. Bees used a highly accurate working memory, remembering which items have already been scanned, resulting in fewer than 1% of re-inspections of items before making a decision. Our results indicate that the small brain of bees, with less parallel processing capacity than mammals, might constrain them to use sequential pattern evaluation even for low quantities.

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.

Factors affecting the measure of inhibitory control in a fish (Poecilia reticulata).

Inhibitory control allows an individual to block automatic responses as well as to control behaviour and attention. There is growing evidence that many species possess this ability, although the difference in performance among species is great. Inhibitory control has been frequently measured using the detour task: a desired reward is placed behind a transparent barrier, and the animal has to inhibit the tendency to directly move toward the goal, instead making a detour around the barrier. Mammals' and birds' inhibitory performance varies according to several factors, such as the distance from the reward and its value, and in dogs, the breed also affects it. We investigated whether these factors affected performance in a fish, the guppy (Poecilia reticulata), by using the detour task, with reaching a social group as goal. We found that guppies were more proficient in making a detour around the barrier when the goal was far, but the value of the reward (i.e., the size of the social group) had no effect. We also found a clear effect of strain, with the guppies that descended from a wild population performing better than the domesticated guppies. Our study revealed that some of the factors affecting inhibitory control in warm-blooded vertebrates also modulate the performance of fish. These factors should be taken into account when comparing this function across species.

Fish perform like mammals and birds in inhibitory motor control tasks.

Inhibitory control is an executive function that positively predicts performance in several cognitive tasks and has been considered typical of vertebrates with large and complex nervous systems such as primates. However, evidence is growing that some fish species have evolved complex cognitive abilities in spite of their relatively small brain size. We tested whether fish might also show enhanced inhibitory control by subjecting guppies, Poecilia reticulata, to the motor task used to test warm-blooded vertebrates. Guppies were trained to enter a horizontal opaque cylinder to reach a food reward; then, the cylinder was replaced by a transparent one, and subjects needed to inhibit the response to pass thought the transparency to reach the food. Guppies performed correctly in 58% trials, a performance fully comparable to that observed in most birds and mammals. In experiment 2, we tested guppies in a task with a different type of reward, a group of conspecifics. Guppies rapidly learned to detour a transparent barrier to reach the social reward with a performance close to that of experiment 1. Our study suggests that efficient inhibitory control is shown also by fish, and that its variation between-species is only partially explained by variation in brain size.

Experimental setting affects the performance of guppies in a numerical discrimination task.

A recent study found that guppies (Poecilia reticulata) can be trained to discriminate 4 versus 5 objects, a numerical discrimination typically achieved only by some mammals and birds. In that study, guppies were required to discriminate between two patches of small objects on the bottom of the tank that they could remove to find a food reward. It is not clear whether this species possesses exceptional numerical accuracy compared with the other ectothermic vertebrates or whether its remarkable performance was due to a specific predisposition to discriminate between differences in the quality of patches while foraging. To disentangle these possibilities, we trained guppies to the same numerical discriminations with a more conventional two-choice discrimination task. Stimuli were sets of dots presented on a computer screen, and the subjects received a food reward upon approaching the set with the larger numerosity. Though the cognitive problem was identical in the two experiments, the change in the experimental setting led to a much poorer performance as most fish failed even the 2 versus 3 discrimination. In four additional experiments, we varied the duration of the decision time, the type of stimuli, the length of training, and whether correction was allowed in order to identify the factors responsible for the difference. None of these parameters succeeded in increasing the performance to the level of the previous study, although the group trained with three-dimensional stimuli learned the easiest numerical task. We suggest that the different results with the two experimental settings might be due to constraints on learning and that guppies might be prepared to accurately estimate patch quality during foraging but not to learn an abstract stimulus-reward association.

Development and testing of a rapid method for measuring shoal size discrimination.

The shoal-choice test is a popular method to investigate quantity discrimination in social fish based on their spontaneous preference for the larger of two shoals. The shoal-choice test usually requires a long observation time (20-30 min), mainly because fish switch between the two shoals with low frequency, thus reducing the possibilities of comparison. This duration limits the use of the shoal-choice test for large-scale screenings. Here, we developed a new version of the shoal-choice test in which the subject was confined in a large transparent cylinder in the middle of the tank throughout the experiment to bound the minimum distance from the stimulus shoals and favour switching. We tested the new method by observing guppies (Poecilia reticulata) in a 4 versus 6 fish discrimination (experiment 1). The new method allowed for a faster assessment of the preference for the larger shoal (<5 min), resulting in potential application for large population screenings. Guppies switched five times more frequently between the two shoals and remained close to the first chosen shoal ten times less compared to experiments with the old method. In experiment 2, we found that with the new method guppies were able to discriminate up to 5 versus 6 fish, a discrimination that was not achieved with the classical method. This last result indicates that minor methodological modifications can lead to very different findings in the same species and suggests that caution should be exercised when interpreting inter-specific differences in quantitative abilities.