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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

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.

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.

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.

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.

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.

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.

Simple Summary Animal models play an important role in research on behaviour and its impairment. Fish larvae allow researchers to conduct experiments on large samples in just a few days and with small-scale experimental infrastructure, substantially increasing research output. However, several aspects of larval biology, including their behaviour, are frequently unknown. Our study has demonstrated that the most important behavioural paradigm for studying anxiety and stress in animals, the open-field test, can be used in the larvae of an important fish genetic model, the medaka. This finding will allow researchers to develop models to study anxiety and stress disorders based on medaka larvae. Abstract 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.

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.

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.

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.

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.

To cope with the variable environment, animals are continuously required to learn novel behaviours or, in certain cases, to inhibit automatic and previously learned behaviours. Traditionally, inhibition has been regarded as cognitively demanding and studied mostly in primates, other mammals and birds, using laboratory tasks, such as the cylinder task. Recent studies have also revealed that fish show high levels of inhibition in the cylinder task. However, conclusions on such results are undermined by evidence that the cylinder task may be inappropriate to compare such phylogenetically distant species. Here, we studied whether a fish, the guppy, Poecilia reticulata, could learn to inhibit behaviour using a different paradigm, which exploited spontaneous foraging behaviour and overcame some drawbacks that characterised the cylinder task. We exposed guppies to live brine shrimp nauplii, Artemia salina, enclosed within a transparent tube. Initially, the guppies attempted to attack the prey but over time showed a rapid decrease of the attacks. Control tests seemed to exclude the possibility that this behavioural trend was due to response to novelty or habituation, and suggested that the guppies were learning to inhibit the foraging behaviour. Memory tests indicated that guppies retained the inhibition of foraging behaviour for at least 24 h. Our study seems to indicate that teleost fish display rapid and durable inhibition of spontaneous foraging behaviour; this may be related to previous evidence, from the cylinder task, supporting efficient behavioural inhibition in this taxon.

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 (

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.

Recognising predators accurately is key to making fine-scale adjustments to behaviour that enhance survival and maximise overall fitness for prey. Prey incorporate information from specific predator features in order to recognise predators and the risk they pose. For olfactory cues, prey can use both predator odour and diet cues to recognise predators. The role of diet cues in predator recognition has only been tested when they provide information about risk and act as an unconditioned stimulus. Thus, it is unclear whether prey use diet cues in the development of more general predator recognition templates. Here, we tested whether diet cues that contain no apparent information about the prey’s vulnerability to the predator are used by prey when they learn to recognise predators. We trained predator-naive wood frog tadpoles (Lithobates sylvaticus) to recognise the odour of a novel crayfish (Orconectes virilis) as risky by pairing tadpole alarm cues with the odour of crayfish fed one of two diets: alfalfa pellets or earthworms (Lumbricus sp.). We tested tadpoles from each group for their response to one of the two crayfish diet odour combinations or a water control. Tadpoles displayed antipredator responses to crayfish odour, irrespective of diet. However, their responses were stronger when tadpoles were exposed to crayfish fed the same diet as during training. Such results demonstrate that diet cues play a previously unrecognised but subtle role in predator recognition and suggest that flexibility in prey choice can lead to an advantage for the predator. Recognising predators and the threat they pose is critical for prey to adjust their behaviour in response to fluctuations in predation risk. There is therefore a need to understand how prey use different cues to develop effective recognition templates that allow for threat-sensitive adjustments to behaviour. Here, we demonstrate that diet cues of predators contribute to the development of predator recognition templates by prey. These results provide new information about how prey develop recognition templates for predators and that, by incorporating diet cues, they are able to adjust their responses to variable risk posed by different predators within a population. Additionally, we suggest that generalist diets may provide unrecognised benefits to predators when switching between prey types.

We investigated the behavioural and learning response of zebrafish larvae to chemicals released by injured conspecifics (the alarm cue). Many aquatic vertebrates and invertebrates exhibit an innate antipredator response to alarm cues because in nature, they reliably indicate the presence of predators. Likewise, when an individual simultaneously perceives a novel odour and alarm cue, it learns to recognise the novel odour as a predator odour. Alarm cue-mediated behavioural response and learning have been reported in some fish and amphibians during early ontogeny , but in zebrafish, they have been described only for adults. In this study, we demonstrated that zebrafish at 12 and 24 days post fertilization exhibited reduced activity when exposed to alarm cue obtained by homogenised larvae of the same age, with this response being greater for the older zebrafish. In addition, we showed that 24-dpf zebrafish conditioned with alarm cue plus a novel odour learned to recognise the novel odour as a threat and responded to it with antipredator behaviour. The innate behavioural response and the learned response after conditioning may be used to develop paradigms with which to study anxiety, fear, stress, learning and memory in zebrafish larvae.

Aquatic prey species show sophisticated mechanisms to adjust their antipredator behaviours to the level of risk, which they estimate either by direct experience with predators or from indirect indicators such as chemical alarm cues released by injured conspecifics. For instance, evidence suggests that the alarm cues of tadpoles exposed to high levels of background predation risk elicit a stronger antipredator response compared to alarm cues of tadpoles exposed to low risk. Similarly, the alarm cues of tadpoles from environments with reduced vegetation cover might cause a stronger response than alarm cues of tadpoles from environments with abundant vegetation because tadpoles suffer high predation when vegetation is scarce. I tested this hypothesis in the edible frog, Pelophylax esculentus, by comparing the response of focal tadpoles (not exposed to vegetation manipulation) to alarm cues of donor tadpoles raised from eggs in either high- or low-vegetation treatment. I also tested the alarm cues of donor tadpoles switched from high- to low-vegetation treatments and vice versa after hatching because this would enable understanding whether an eventual difference in alarm cues occurred due to the embryonic or larval environments and whether the treatments at the two developmental stages had interactive effects. Alarm cues from the low-vegetation, and thus the high-risk, treatment elicited stronger antipredator response in focal tadpoles in comparison to the alarm cues from the high-vegetation, low-risk treatment. Results from switching donor tadpoles between vegetation treatments after hatching suggested that the observed effect was due to the vegetation treatment experienced by donor tadpoles during the larval stage, with no interactive effects. Chemical alarm cues convey information about cover abundance, an environmental factor that indirectly covaries with predation risk.

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

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.

Evidence from a growing number of organisms suggests that individuals show consistent performance differences in cognitive tasks. According to empirical and theoretical studies, these cognitive differences might be at least partially related to personality. We tested this hypothesis in the guppy, Poecilia reticulata, by comparing individuals with different degree of sociability in the discrimination of shoals formed by a different number of conspecifics. We found that individual guppies show repeatability of sociability as expected for personality traits. Furthermore, individuals with higher sociability showed poorer shoal size discrimination performance and were less efficient in choosing the larger shoal compared to individuals with low sociability. As choosing the larger shoal is an important strategy of defense against predators for guppies, we discuss this relationship between personality and cognition in the light of its fitness consequences.

The ability to discriminate between different quantities is useful to an animal since it allows adaptive decisions in several contexts. In humans, some individuals are generally better at solving different numerical and mathematical problems, but we currently ignore whether other species also show individual differences in quantitative abilities across different contexts. Aiming to investigate this possibility, we observed the performances of individual guppies (Poecilia reticulata) in an antipredator task that required the discrimination between shoals made of a different number of conspecifics (4 versus 6 fish) and, successively, in two foraging tasks that required the discrimination between different numbers of food items (4 versus 6 items) and food items with different sizes (0.67 ratio between the two surface areas). Overall, guppies achieved comparable performances in the discrimination of shoal size and food item size, but showed reduced accuracy in the discrimination between different numbers of food items. Performances of guppies were correlated between the discrimination of shoal size and food item size, suggesting the existence of individual differences in quantity discrimination abilities in different contexts in this species. Unexpectedly, this relationship was negative, and some guppies were better in the antipredator task, while others were better in the foraging task. Our results agree with the general trend of the literature on animals that suggests the existence of multiple cognitive functions whose activation are task specific, and also introduce the possibility that some of these functions are subject to constraints. In this work, we provided the first clear evidence of individual differences in the ability to solve quantity discriminations in a fish. Guppies were observed in three quantity discrimination tasks: recognising and joining the larger shoal of conspecifics among two options, a behaviour that confers protection from predators; selecting the larger number of food items between two sets; and selecting the food item with larger size. Guppies that were more accurate in the shoal size discrimination achieved lower scores in the food item size discrimination. Some individuals gain advantage from cognitive abilities that confer defence against predators, while other individuals from those abilities that improve foraging success.

Mammals and birds can process ordinal numerical information which can be used, for instance, for recognising an object on the basis of its position in a sequence of similar objects. Recent studies have shown that teleost fish possess numerical abilities comparable to those of other vertebrates, but it is unknown if they can also learn ordinal numerical relations. Guppies (Poecilia reticulata) learned to recognise the 3rd feeder in a row of 8 identical ones even when inter-feeder distance and feeder positions were varied among trials to prevent the use of any spatial information. To assess whether guppies spontaneously use ordinal or spatial information when both are simultaneously available, fish were then trained with constant feeder positions and inter-feeder distance. In probe trials where these two sources of information were contrasted, the subjects selected the correct ordinal position significantly more often than the original spatial position, indicating that the former was preferentially encoded during training. Finally, a comparison between subjects trained on the 3rd and the 5th position revealed that guppies can also learn the latter discrimination, but the larger error rate observed in this case suggests that 5 is close to the upper limit of discrimination in guppies.