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In nature, animals are exposed to stressors that occur with different likelihood throughout the day, such as risk of predation and human disturbance. Hence, the stress response is expected to vary plastically to adaptively match these challenges. Several studies have supported this hypothesis in a wide range of vertebrate species, including some teleost fish, mostly through evidence of circadian variation in physiology. However, in teleost fish, circadian variation in behavioural stress responses is less understood. Here, we investigated the daily rhythm of stress response at the behavioural level in the zebrafish Danio rerio. We exposed individuals and shoals to an open field test every 4 h over a 24 h cycle, recording three behavioural indicators of stress and anxiety levels in novel environments (thigmotaxis, activity and freezing). Thigmotaxis and activity significantly varied throughout the day with a similar pattern, in line with a stronger stress response in the night phase. The same was suggested by analysis of freezing in shoals, but not in individual fish, in which variation appeared mostly driven by a single peak in the light phase. In a control experiment, we observed a set of subjects after familiarisation with the open- field apparatus. This experiment indicated that activity and freezing might present a daily rhythmicity that is unrelated to environmental novelty, and thus to stress responses. However, the thigmotaxis was constant through the day in the control condition, suggesting that the daily variation of this indicator is mostly attributable to the stress response. Overall, this research indicates that behavioural stress response of zebrafish does follow a daily rhythm, although this may be masked using behavioural indicators other than thigmotaxis. This rhythmicity can be relevant to improve welfare in aquaculture and reliability of behavioural research in fish models

Living in a social group may impose cognitive demands, for example individual recognition, social memory and the inhibition of behaviour when it is not adaptive. As the neural substrates for these cognitive skills are metabolically expensive, cognitive abilities may be positively related to the complexity of the social system. Where there is large spatiotemporal variation in the ecological conditions experienced and hence in the social system exhibited by species, selection may favour adaptive phenotypic plasticity of cognitive abilities involved in social tasks rather than evolved differences across populations. Here, we tested this hypothesis in a social-living teleost fish, the guppy, Poecilia reticulata. We exposed new-born guppies to treatments that altered two parameters of social environmental complexity: group size (experiment 1) and group stability (experiment 2). Then, we assessed guppies' inhibitory control, the ability to withhold responding to a stimulus, a cognitive function that is critically involved in social interactions. In experiment 1, guppies reared alone showed higher levels of behavioural inhibition in a foraging task compared to guppies reared in pairs or in groups of six. In addition, we found that individuals' variance in performance was smaller for fish raised alone. In experiment 2, guppies reared in a stable social group showed greater inhibition than those from groups subjected to frequent fission–fusion events. These results reveal phenotypic plasticity of inhibitory control in guppies; however, contrary to prediction, they indicate greater inhibitory abilities developing in individuals exposed to ‘simpler’ social environments.

Brain-derived neurotropic factor (BDNF) has established roles in neuronal survival and differentiation, in synaptic plasticity, and in neurotransmitters release. Several lines of evidence suggest that variations in BDNF might alter behaviour and contribute to neurobehavioural disorders. We investigated the functional effects of BDNF loss on behaviour by phenotyping a recently-generated CRISPR/Cas9 bdnf

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.

In mammals and birds, interindividual variability in inhibitory control is often explained by developmental changes and personality. Teleost fish exhibit one of the highest levels of intraspecific variability in inhibitory control described in vertebrates, but the underlying causes have not been fully described. We investigated the effects of development and personality on inhibitory control of a teleost fish, the guppy. In experiment 1, we compared the performance of 10-, 20-, 40- and 90-day-old guppies in the cylinder task, which requires subjects to inhibit the tendency to swim towards a food reward. We found that guppies of all ages tested were capable of executing the motor inhibition required by the task. However, there was a U-shaped effect of age on performance: 10- and 90-day-old guppies had lower inhibitory control compared to 20- and 40-day-old guppies. In experiment 2, we correlated guppies’ inhibitory control performance with three tests aimed to assess different personality trials: boldness (scototaxis test), exploration (open-field test) and sociability (mirror test). We found a significant covariation only for the open-field test, which might indicate that more explorative guppies exhibited greater inhibitory control. Overall, this study suggests that ontogeny and personality may have a role in determining interindividual differences in a teleost fish similar to that described in mammals and birds.

Abstract Research on several social fishes has revealed that shoals constituted by familiar individuals behave remarkably differently compared to shoals formed by unfamiliar individuals. However, whether these behavioural changes may arise also in shoals composed by a mixture of familiar and unfamiliar individuals, a situation that may commonly occur in nature, is not clear. Here, we observed the behaviour of Mediterranean killifish (Aphanius fasciatus) shoals that were composed by both familiar and unfamiliar individuals (i.e. individuals were familiar to each other in pairs) and compared it with shoals entirely made by either unfamiliar or familiar individuals. Shoals formed by familiar individuals took longer to emerge from a refuge and swam more cohesively compared to shoals formed by unfamiliar fish. Shoals formed by a mixture of familiar and unfamiliar individuals behaved as shoals formed by unfamiliar individuals. Moreover, mixed shoals did not segregate in pairs according to their familiarity. This study suggests that mixed shoals do not show the behavioural effects of familiarity. Significance statement Laboratory studies have compared the behaviour of shoals formed by familiar fish versus shoals formed by unfamiliar fish, finding notable advantages in the former ones, such as improved antipredator and foraging behaviour. However, comparing these two opposite shoal types may not provide information on the natural situation, because in nature, shoals often change composition. We investigated how shoals formed by a mixture of familiar and unfamiliar fish behaved. We analysed shoals’ preference for open environment versus covers and shoals’ swimming cohesion. Results showed that shoals formed by both familiar and unfamiliar individuals mostly behave like shoals entirely formed by unfamiliar individuals. This suggests that the advantages of social groups formed by familiar fish might be hardly seen in nature for species in which shoal composition changes frequently.

Parabens are classified as endocrine disrupting chemicals due to their ability to activate several nuclear receptors causing changes in hormones-dependent signalling pathways. Central nervous system of developing organisms is particularly vulnerable to changes in hormonal pathways, which could lead to altered brain function, abnormal behaviour and even diseases later in life. The aim of the present study was to investigate the effects of exposure to butylparaben (BuP), ethylparaben (EtP) and methylparaben (MeP) during early development on nervous system using zebrafish larvae's behavioural models. Zebrafish were exposed until 4 days post fertilization (dpf) to three concentrations of each paraben chosen considering the environmentally realistic concentrations of human exposure and the benchmark-dose lower bound calculated for zebrafish larvae (BuP: 5, 50 and 500 μg/L; EtP: 50, 500 and 5000 μg/L; MeP: 100, 1000 and 10,000 μg/L). Activity in novel and in familiar environment, thigmotaxis, visual startle response and photic synchronization of the behavioural circadian rhythms were analysed at 4, 5 and 6 dpf. Zebrafish larvae exposed to BuP 500 μg/L and EtP 5000 μg/L revealed increased anxiety-like behaviour in novel environment. Larvae treated with 500 μg/L of BuP showed reduced activity in familiar and marginally in unfamiliar environment, and larvae exposed to 5000 μg/L of EtP exhibited hyperactivity in familiar environment. Parabens exposure did not influence the visual startle response and the photic synchronization of circadian rhythms in zebrafish larvae. This research highlighted as the exposure to parabens has the potential to interfere with behavioural development of zebrafish.

Triclocarban (TCC), is an antimicrobial component in personal care products and it is one of the emerging contaminants since it has been detected in various environmental matrices. Its presence in human cord blood, breast milk, and maternal urine raised issues about its possible impact on development and increased concerns about the safety of daily exposure. This study aims to provide additional information about the effects of zebrafish early-life exposure to TCC on eye development and visual function. Zebrafish embryos were exposed to two concentrations of TCC (5 and 50 μg/L) for 4 days. TCC-mediated toxicity was assessed in larvae at the end of exposure and in the long term (20 days post fertilization; dpf), through different biological end-points. The experiments showed that TCC exposure influences the retinal architecture. In 4 dpf treated larvae, we found a less organized ciliary marginal zone, a decrease in the inner nuclear and inner plexiform layers, and a decrease in the retinal ganglion cell layer. Photoreceptor and inner plexiform layers showed an increase in 20 dpf larvae at lower and both concentrations, respectively. The expression levels of two genes involved in eye development (mitfb and pax6a) were both decreased at the concentration of 5 μg/L in 4 dpf larvae, and an increase in mitfb was observed in 5 μg/L-exposed 20 dpf larvae. Interestingly, 20 dpf larvae failed to discriminate between visual stimuli, demonstrating notable visual perception impairments due to compound. The results prompt us to hypothesize that early-life exposure to TCC may have severe and potentially long-term effect on zebrafish visual function.

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.

Lateralization of cognitive functions impacts many behaviours related to fitness and, in most species, varies greatly among individuals. Laboratory and field studies have suggested that within-species variation in lateralization is partly due to phenotypic plasticity. For example, in fish, prey that have experienced predation risk during early ontogeny develop highly lateralized phenotypes, and this lateralization often favours prey in evading predators. In contexts other than predation, plasticity of lateralization has also been reported for adult fish. Therefore, we asked whether adult fathead minnows, Pimephales promelas, exposed to high predation risk would also show plasticity linked to increase lateralization. We exposed minnows to conspecific alarm cues for up to 8 days to simulate predation risk and tested their lateralization with a standard detour test. The treatment affected lateralization but in an unexpected direction: Individuals exposed to high predation risk showed lower lateralization scores compared to control fish. In addition, fish within groups exposed to risk reduced the variability in their directionality of lateralization; that is, they showed a similar turning preference in the detour task. Our study suggests that lateralization can vary in response to predation risk in adult fish. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

An optimal foraging strategy often requires identifying and choosing the larger amount of food in the presence of multiple options, in order to maximize food intake. Food quantity estimation frequently depends on the perceptual ability to segregate food from the surrounding background. In human and nonhuman animals, it has recently been shown that the perception of food size is affected by the size of the background on which food is presented, with a tendency to overestimate food items encircled by a small background. This perceptual bias resembles an illusory effect known as the Delboeuf illusion. We investigated whether this bias occurs in five fish species: zebrafish, Danio rerio, redtail splitfin, Xenotoca eiseni, angelfish, Pterophyllum scalare, Siamese fighting fish, Betta splendens, and three-spot gourami, Trichopodus trichopterus. In control trials, we observed their spontaneous preference for choosing the larger of two amounts of food. In test trials, two same-sized food amounts were presented on a small and a large plate, a pattern that causes the Delboeuf illusion in humans. Similarities in subjects’ choices between the five species were greater than differences, with a significant bias towards selecting the food amount on the large plate. This suggests the existence of a perceptual bias, although one operating in the opposite direction from that of humans, which might interfere with the accurate food size estimation necessary for an optimal foraging strategy.

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.

Teleosts display the highest level of brain plasticity of all vertebrates. Yet we still know little about how seasonality affects fish behaviour and the underlying cognitive mechanisms since the common neurobehavioral fish models are native to tropical environments where seasonal variation is absent or reduced. The medaka, Oryzias latipes, which inhabits temperate zone habitats, represents a promising model in this context given its large phenotypic changes associated with seasonality and the possibility to induce seasonal plasticity by only manipulating photoperiod. Here, we report the first extended investigation of seasonal plasticity in medaka behaviour and cognition, as well as the potential underlying molecular mechanisms. We compared medaka exposed to summer photoperiod (16 h light:8 h dark) with medaka exposed to winter photoperiod (8 h light:16 h dark), and detected substantial differences. Medaka were more active and less social in summer photoperiod conditions, two effects that emerged in the second half of an open-field and a sociability test, respectively, and might be at least in part related to habituation to the testing apparatus. Moreover, the cognitive phenotype was significantly affected: in the early response to a social stimulus, brain functional lateralisation shifted between the two hemispheres under the two photoperiod conditions, and inhibitory and discrimination learning performance were reduced in summer conditions. Finally, the expression of genes encoding key pituitary hormones, tshß and gh, and of the tshß regulatory transcription factor tef in the brain was increased in summer photoperiod conditions. This work reveals remarkable behavioural and cognitive phenotypic plasticity in response to photoperiod in medaka, and suggests a potential regulatory role for the same hormones involved in seasonal plasticity of other vertebrates.

Simple Summary Environmental enrichment is extremely important for an individual’s neural, cognitive and behavioral development. Emerging animal models, such as teleost fish, may contribute to our understanding of enrichment-driven cognitive plasticity. We studied the cognitive consequences of living in enriched conditions in Poecilia reticulata. In particular, we compared subjects raised alone in a barren aquarium versus subjects exposed to enriched aquaria (with conspecifics, natural substrate, plants, and live prey) in three different cognitive tasks to measure learning, inhibitory control, and cognitive flexibility. Our results showed that guppies from the enriched aquaria learned a color discrimination faster compared to the subject raised in barren conditions. However, in the two remaining cognitive tasks, we found no effect from the treatment, suggesting that enrichment does not affect inhibitory control and cognitive flexibility. This study reveals that enrichment-driven plasticity affects only specific cognitive abilities. Abstract Many aspects of animal cognition are plastically adjusted in response to the environment through individual experience. A remarkable example of this cognitive phenotypic plasticity is often observed when comparing individuals raised in a barren environment to individuals raised in an enriched environment. Evidence of enrichment-driven cognitive plasticity in teleost fish continues to grow, but it remains restricted to a few cognitive traits. The purpose of this study was to investigate how environmental enrichment affects multiple cognitive traits (learning, cognitive flexibility, and inhibitory control) in the guppy, Poecilia reticulata. To reach this goal, we exposed new-born guppies to different treatments: an enrichment environment with social companions, natural substrate, vegetation, and live prey or a barren environment with none of the above. After a month of treatment, we tested the subjects in a battery of three cognitive tasks. Guppies from the enriched environment learned a color discrimination faster compared to guppies from the environment with no enrichments. We observed no difference between guppies of the two treatments in the cognitive flexibility task, requiring selection of a previously unrewarded stimulus, nor in the inhibitory control task, requiring the inhibition of the attack response toward live prey. Overall, the results indicated that environmental enrichment had an influence on guppies’ learning ability, but not on the remaining cognitive functions investigated.

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.

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.

Cognitive sex differences have been reported in several vertebrate species, mostly in spatial abilities. Here, I review evidence of sex differences in a family of general cognitive functions that control behaviour and cognition, i.e., executive functions such as cognitive flexibility and inhibitory control. Most of this evidence derives from studies in teleost fish. However, analysis of literature from other fields (e.g., biomedicine, genetic, ecology) concerning mammals and birds reveals that more than 40% of species investigated exhibit sex differences in executive functions. Among species, the direction and magnitude of these sex differences vary greatly, even within the same family, suggesting sex-specific selection due to species' reproductive systems and reproductive roles of males and females. Evidence also suggests that sex differences in executive functions might provide males and females highly differentiated cognitive phenotypes. To understand the evolution of cognitive sex differences in vertebrates, future research should consider executive functions.

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.

The study of swimming behavior is an important part of fish biology research and the swim tunnel is used to study swimming performance as well as metabolism of fish. In this investigation, we have developed a user-friendly, automated, modular, and low-cost swim tunnel that permits to study the performance of one or more fish separately, as well as a small group of individuals. To validate our swim tunnel, we assessed swimming activity of four different species (zebrafish, medaka, guppy, and cavefish) recording reliable data of swimming behavior and performance. Because swimming behavior has been recently used in different fields from physiology to ecotoxicology, our setup could help researchers with a low-cost solution.

Behavioural innovations allow an individual to solve new problems or find new solutions to an existing problem. Despite being considered an important source of phenotypic plasticity and evolutionary changes, innovative problem-solving remains poorly understood, except in a few species of mammals and birds. We investigated innovative problem-solving performance and its underlying psychological mechanisms in a teleost fish, the guppy Poecilia reticulata. We assayed guppies in Thorndike’s puzzle-box problem: we placed them in a small chamber, where they had to learn to dislodge an object to access a tunnel leading to their home tank. Guppies showed heightened performance with most individuals (23 out of 24) solving the problem, within, on average, three trials. After a fish solved the task for the first time, improvement was still visible in the form of an increased likelihood to solve the problem over trials. An individual’s sex and willingness to solve the task were unimportant, but behavioural traits related to neophilia significantly predicted problem-solving performance. High exploration in a new environment and high attraction towards novel objects favoured the guppies in learning the task solution. Our finding suggested that this fish species shows remarkable performance and individuality in innovative problem-solving. As observed in warm-blooded vertebrates, these cognitive features may have important consequences for individual fitness and the species’ invasiveness in nature. Behavioural innovation is an important mechanism that allows animals to adapt to their environment. Among the others, it permits solving new problems and obtaining new resources. However, innovative problem-solving remains poorly understood except for a few species of mammals and birds. Our study suggests that fish can learn a novel problem-solving task and improve over time to find the solution. Fish learned to dislodge an object to access a tunnel leading to their home tank. Problem-solving performance was linked to the propensity to explore novel objects and novel environments. Individuals with greater neophilia may have higher propensities for innovative problem-solving.

Individual fitness often depends on the ability to inhibit behaviours not adapted to a given situation. However, inhibitory control can vary greatly between individuals of the same species. We investigated a mechanism that might maintain this variability in zebrafish ( Danio rerio ). We demonstrate that inhibitory control correlates with cerebral lateralization, the tendency to process information with one brain hemisphere or the other. Individuals that preferentially observed a social stimulus with the right eye and thus processed social information with the left brain hemisphere, inhibited foraging behaviour more efficiently. Therefore, selective pressures that maintain lateralization variability in populations might provide indirect selection for variability in inhibitory control. Our study suggests that individual cognitive differences may result from complex multi-trait selection mechanisms.

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.

Black Soldier Fly (BSF) meal is considered as an alternative, emerging and sustainable ingredient for aquafeed production. However, results on fish physiological responses are still fragmentary and often controversial, while no studies are available on fish behavior in response to these new diets. The present work represents the first comprehensive multidisciplinary study aimed to investigate zebrafish physiological and behavioural responses to BSF-based diets. Five experimental diets characterized by increasing inclusion levels (0, 25, 50, 75 and 100% respect to fish meal) of full fat BSF prepupae meal were tested during a 2-months feeding trial. Prepupae were cultured on coffee silverskin growth substrate enriched with a 10% Schizochytrium sp. to improve insects’ fatty acids profile. The responses of zebrafish were assayed through biometric, histological, gas chromatographic, microbiological, spectroscopic, molecular and behavioural analyses. Results evidenced that BSF-based diets affected fish fatty acid composition, while behavioural tests did not show differences among groups. Specifically, a 50% BSF inclusion level diet represented the best compromise between ingredient sustainability and proper fish growth and welfare. Fish fed with higher BSF inclusions (75 and 100%) showed hepatic steatosis, microbiota modification, higher lipid content, fatty acid modification and higher expression of immune response markers.

The use of juvenile and larval fish models has been growing in importance for several fields. Accordingly, the evaluation of behavioural tests that can be applied to larvae and juveniles is becoming increasingly important. We tested medaka at four different ages (1, 10, 30, and 120 dph) in the open field test, one of the most commonly used behavioural assays, to investigate its suitability for larvae and juveniles of this species. We also explored ontogenetic variation in behaviour during this test. On average, adult 120-day-old medaka showed higher locomotor activity in terms of distance moved compared with younger fish. Our analysis suggests that this effect was derived from both quantitative changes in locomotion related to the ontogenetic increase in fish size as well as qualitative changes in two aspects of locomotor behaviour. Specifically, time spent moving was similar between 1- and 10-day-old medaka, but progressively increased with development. In addition, we revealed that adult medaka showed constant levels of activity, whereas younger medaka progressively reduced their activity over the course of the entire experiment. The thigmotaxis behaviour typically used to assess anxiety in the open field test emerged at 120 days post-hatching, even though a difference in the temporal pattern of spatial preference emerged earlier, between 10 and 30 days post-hatching. In conclusion, some measures of the open field test such as total distance moved allow behavioural phenotyping in the medaka of all ages, although with some degree of quantitative and qualitative developmental variation. In contrast, immature medaka appear not to exhibit thigmotactic 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.

In a number of animal species, individuals differ in their ability to solve cognitive tasks. However, the mechanisms underlying this variability remain unclear. It has been proposed that individual differences in cognition may be related to individual differences in behavior (i.e., personality); a hypothesis that has received mixed support. In this study, we investigated whether personality correlates with the cognitive ability that allows inhibiting behavior in 2 teleost fish species, the zebrafish Danio rerio and the guppy Poecilia reticulata. In both species, individuals that were bolder in a standard personality assay, the open-field test, showed greater inhibitory abilities in the tube task, which required them to inhibit foraging behavior toward live prey sealed into a transparent tube. This finding reveals a relationship between boldness and inhibitory abilities in fish and lends support to the hypothesis of a link between personality and cognition. Moreover, this study suggests that species separated by a relatively large phylogenetic distance may show the same link between personality and cognition, when tested on the same tasks.

Once considered a human characteristic, the presence of correlations between individuals' performance in cognitive tasks has now been reported in a range of vertebrates. In humans, an important source of cognitive variability is inhibitory control: some individuals are consistently more efficient in inhibitory tasks and this affects individual differences in other cognitive tasks, including measures of general intelligence. We looked for these two types of individual differences in a teleost fish, the guppy Poecilia reticulata. First, we observed guppies in two inhibitory control tasks. In the tube task, guppies had to inhibit the tendency to attack live prey sealed into a transparent tube. In the cylinder task, guppies had to inhibit the tendency to swim directly toward a food item placed inside a transparent cylinder and rather detour and enter the cylinder from the open sides. Individual rank performance was maintained between the two inhibitory tasks, suggesting individual differences in inhibitory control across tasks in this species. Then, we tested the same set of guppies in a problem-solving task, whereby they had to learn to dislodge an object that prevented the access to a food reward. Neither the tube task nor the cylinder task score predicted guppies' problem-solving performance. Our study demonstrates that fish exhibit consistent individual differences in inhibitory control, as expected if this trait has a common evolutionary origin in vertebrates. Yet, in fish, these individual differences appear not to be related to other cognitive processes such as those required for problem solving.

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.

We assessed whether zebrafish, Danio rerio, display inhibitory control using a simple and rapid behavioural test. Zebrafish were exposed to a prey stimulus placed inside a transparent tube, which initially elicited attack behaviour. However, zebrafish showed a rapid reduction in the number of attacks towards the prey, which indicated the ability to inhibit their foraging behaviour. Zebrafish also exhibited mnemonic retention of foraging inhibition, as indicated by a reduced number of attacks in a subsequent exposure to the unreachable prey. The ability to inhibit the foraging behaviour varied across three genetically separated wild-type strains and across different individuals within strains, suggesting that zebrafish show heritable within-species differences in inhibitory control. Our behavioural test might be suitable for screening large zebrafish populations in mutational studies and assessing the effects of pharmacologically active substances on inhibitory control.

Summary Day length in conjunction with seasonal cycles affects many aspects of animal biology. We have studied photoperiod-dependent alterations of complex behavior in the teleost, medaka (Oryzias latipes), a photoperiodic breeder, in a learning paradigm whereby fish have to activate a sensor to obtain a food reward. Medaka were tested under a long (14:10 LD) and short (10:14 LD) photoperiod in three different groups: mixed-sex, all-males, and all-females. Under long photoperiod, medaka mixed-sex groups learned rapidly with a stable response. Unexpectedly, males-only groups showed a strong learning deficit, whereas females-only groups performed efficiently. In mixed-sex groups, female individuals drove group learning, whereas males apparently prioritized mating over feeding behavior resulting in strongly reduced learning performance. Under short photoperiod, where medaka do not mate, male performance improved to a level similar to that of females. Thus, photoperiod has sex-specific effects on the learning performance of a seasonal vertebrate., Graphical abstract, Highlights • Fish can learn by association using a self-feeder system and food reward • Medaka show sex-specific and seasonal differences in cognitive performance • On long days, sexually active male medaka are poor learners compared with females • Under short days, non-mating male medaka learn better, Ichthyology; Cognitive neuroscience

As a consequence of sexual selection, males and females may exhibit wide behavioural differences, for example, spatial behaviour. In fish, the two sexes often show different exploratory tendencies. This sex difference has been usually studied by testing individual fish. As many fish species live in social groups with different sex composition, the aforementioned approach might not picture the natural variation of the exploratory behaviour expressed by males and females. Here, we observed shoals of four Mediterranean killifish, Aphanius fasciatus, with three different sex compositions (4 females, 4 males, or 2 females and 2 males) during the exploration of a novel environment. Sex composition of the shoals did not predict the latency to emerge from a shelter into the novel environment. However, once emerged, shoals composed by four males displayed reduced exploratory behaviour compared to 4-female and mixed-sex shoals. These results indicated that sex differences in exploration subsist also at group level and highlighted the importance of sex composition in determining the behaviour of the entire shoal.

Many fish species are social, and individuals spend most of their lives in shoals, but sociability can vary greatly between species, populations and individuals. Sociability has been largely studied by measuring the time spent by a focal fish in proximity to one or more conspecifics. To control for the behaviour of the stimulus fish, the conspecifics have often been substituted by a mirror, on the assumption that the subject perceives its mirror image as a conspecific. The validity of the mirror test has recently been questioned, at both the behavioural and the molecular level, because of the discrepancy in fish responses when exposed to a mirror image and to a live conspecific. In this study, we compared the sociability scores of a social fish, the guppy, Poecilia reticulata, obtained using live fish or a mirror as a stimulus, to assess the validity of the mirror test. We found that the sociability score assessed using the standard mirror test was not significantly correlated with the sociability assessed using live stimuli. Nevertheless, we observed a positive correlation between the scores of the two tests when the mirror test was performed in a more naturalistic context, controlling for the minimum distance between the stimulus fish and the mirror. Our findings provide evidence of the reliability of the mirror test as a measure of sociability compared to the test using live conspecifics when certain requirements are satisfied.

Living in a group offers the chance to follow the choices and the behaviours of other individuals. Following a group mate might confer fitness advantages if the group mate knows about resources such as food or shelters. Shoaling fish often follow larger group mates which, in most species, are generally older and therefore more experienced. Yet, the effect of individuals' characteristics other than size on following behaviour remains to be understood. For example, familiar fish and female shoals have been reported as more cohesive, which might be due to a differential tendency to follow in relation to familiarity and sex. Here, we investigated whether size, familiarity, sex, and the interaction between these factors affect fish following behaviour. We observed pairs of differently sized Mediterranean killifish, Aphanius fasciatus, exploring a new environment, and we recorded whether the rear fish followed the front fish when the latter changed swimming direction. In female and male pairs, and in unfamiliar pairs, smaller fish were more likely to follow the directional change of the larger fish than vice versa. In mixed-sex pairs and in familiar pairs, however, size did not affect following behaviour and larger fish followed as much as smaller fish did. Our results revealed that killifish's following decisions are determined by the size of the individuals, their level of familiarity, and their sex. These characteristics may have a notable impact on the behaviour of fish groups in nature.

Animal species differ considerably in their ability to detour around a see-through obstacle to reach a goal positioned behind it. This variation is commonly assumed to derive from interspecific differences in the cognitive functions involved in the execution of the task, such as spatial abilities and inhibitory motor control. A teleost fish, the guppy, Poecilia reticulata, has recently been found to outperform many mammals and birds in this type of task. To determine whether this is a typical condition in teleost fish or whether detour abilities vary among fish species as observed in mammals and birds, we compared four distantly related teleosts in a transparent barrier task using a group of conspecifics as the goal. The scores of three species (Poecilia reticulata, Xenotoca eiseni, Oryzias sarasinorum) were similar to those previously reported for fish. The remaining species, the zebrafish, Danio rerio, showed a much higher performance, close to that of warm-blooded animals with the highest scores (e.g. corvids and monkeys). In comparative cognition studies, contextual variables rather than differences in cognitive ability may be responsible for observed differences between species. In a second experiment, we found that the four species were similarly gregarious, excluding a different motivation to reach the target as an explanation for the different performance. In another experiment, however, we found evidence that the zebrafish's higher detour performance might be due to a sensory advantage. Zebrafish used olfactory cues (towards which the barrier was opaque) to navigate to the social stimuli, whereas the guppy, which we used as a control species, preferentially relied on visual information. This study highlights the importance of sensory differences as a source of potential experimental confound in comparative cognition research.

Behavioural responses to the environment often require the suppression of strong internal predispositions or the overriding of external lures, tasks performed by a cognitive function called inhibitory control. Inhibitory control of nonhuman animals is generally measured with the cylinder task: subjects are presented with food inside a transparent cylinder and must inhibit their tendency to reach the food directly and instead detour round the cylinder to solve the task. However, several studies have raised concerns about the validity of this test to compare different species. Recently, a tiny teleost fish, the guppy, Poecilia reticulata, was tested with the cylinder task and scored higher than many mammals and birds. Before considering that guppies display unpredictably high inhibitory control, it is important to exclude that they were advantaged by some noncognitive factors. In particular, guppies could enjoy three advantages: experience with transparent surfaces (i.e. the walls of maintenance aquaria), experience with detouring round see-through obstacles (vegetation in the maintenance aquaria) and the spread of food odour in the water from the sides of the cylinder. We tested whether these factors affect guppies' performance in the cylinder task by manipulating both their experience with transparent surfaces before the task and the diffusion of food odour cues from the cylinder. Guppies raised in transparent aquaria or with transparent panels placed inside the tank did not show advantages over guppies with no experience with transparent surfaces. Furthermore, the guppies’ performance was not reduced when the cylinder was pierced in the middle, so that both visual and olfactory cues lured them in the same direction. These results seem to exclude methodological explanations for the high inhibitory control score of guppies, and they indicate that even teleost fish can display efficient inhibitory control.

Animal species are expected to evolve specialised cognitive abilities to solve the tasks that are critical for their fitness. The literature contains several examples of specialised cognitive abilities, but few regard fish. The guppy, Poecilia reticulata, is a freshwater fish in which females choose their mates based on colouration, and orange‐coloured fruits are important diet enrichments for both sexes. For these reasons, we expect that this species has evolved enhanced learning abilities in colour discrimination compared to other types of discrimination. The comparison between studies in which guppies were tested for colour discrimination and studies in which guppies were tested for shape discrimination seems to support this hypothesis, but direct testing is still lacking. We experimentally compared the learning performance of guppies trained in a red–yellow colour discrimination learning task and that of guppies trained in a shape discrimination learning task using the same, automated conditioning procedure. Guppies trained in the colour discrimination showed greater learning performance, which provides support to the hypothesis that guppies possess enhanced colour discrimination abilities. Moreover, we found that male guppies performed better than females in both shape and colour discrimination learning.