Environmental conditions shape learning in larval zebrafish.

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

One-trial odour recognition learning and its underlying brain areas in the zebrafish.

Distinguishing familiar from novel stimuli is critical in many animals' activities, and procedures based on this ability are among the most exploited in translational research in rodents. However, recognition learning and the underlying brain substrates remain unclear outside a few mammalian species. Here, we investigated one-trial recognition learning for olfactory stimuli in a teleost fish using a behavioural and molecular approach. With our behavioural analysis, we found that zebrafish can learn to recognise a novel odour after a single encounter and then, discriminate between this odour and a different one provided that the molecular structure of the cues is relatively differentiated. Subsequently, by expression analysis of immediate early genes in the main brain areas, we found that the telencephalon was activated when zebrafish encountered a familiar odour, whereas the hypothalamus and the optic tectum were activated in response to the novel odour. Overall, this study provided evidence of single-trial spontaneous learning of novel odours in a teleost fish and the presence of multiple neural substrates involved in the process. These findings are promising for the development of zebrafish models to investigate cognitive functions.

Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

Seeking temporal refugia to heat stress: increasing nocturnal activity despite predation risk.

Flexibility in activity timing may enable organisms to quickly adapt to environmental changes. Under global warming, diurnally adapted endotherms may achieve a better energy balance by shifting their activity towards cooler nocturnal hours. However, this shift may expose animals to new or increased environmental challenges (e.g. increased predation risk, reduced foraging efficiency). We analysed a large dataset of activity data from 47 ibex (Capra ibex) in two protected areas, characterized by varying levels of predation risk (presence versus absence of the wolf-Canis lupus). We found that ibex increased nocturnal activity following warmer days and during brighter nights. Despite the considerable sexual dimorphism typical of this species and the consequent different predation-risk perception, males and females demonstrated consistent responses to heat in both predator-present and predator-absent areas. This supports the hypothesis that shifting activity towards nighttime may be a common strategy adopted by diurnal endotherms in response to global warming. As nowadays different pressures are pushing mammals towards nocturnality, our findings emphasize the urgent need to integrate knowledge of temporal behavioural modifications into management and conservation planning.

Embryonic and larval exposure to propylparaben induces developmental and long-term neurotoxicity in zebrafish model.

Parabens are preservatives found in cosmetics, processed foods, and medications. The harmful repercussions on the central nervous system by one of the most common parabens, propylparaben (PrP), are yet unknown, especially during development. In this study, the neurodevelopmental effects of PrP and long-term neurotoxicity were investigated in the zebrafish model, using an integrated approach. Zebrafish embryos were exposed to two different concentrations of PrP (10 and 1000 µg/L), then larvae were examined for their behavioral phenotypes (open-field behavior, startle response, and circadian rhythmicity) and relevant brain markers (cyp19a1b, pax6a, shank3a, and gad1b). Long-term behavioral and cognitive impacts on sociability, cerebral functional asymmetry and thigmotaxis were also examined on juveniles at 30 dpf and 60 dpf. Moreover, proteomics and gene expression analysis were assessed in brains of 60 dpf zebrafish. Interestingly, thigmotaxis was decreased by the high dose in larvae and increased by the low dose in juveniles. The expression of shank3a and gad1b genes was repressed by both PrP concentrations pointing to possible effects of PrP on neurodevelopment and synaptogenesis. Proteomics analysis evidenced alterations related to brain development and lipid metabolism. Overall, the results demonstrated that early-life exposure to PrP promotes developmental and persistent neurobehavioral alterations in the zebrafish model, affecting genes and protein levels possibly associated with brain diseases.

Embryonic exposure to artificial light at night impairs learning abilities and their covariance with behavioural traits in teleost fish.

The natural light cycle has profound effects on animals' cognitive systems. Its alteration owing to human activities, such as artificial light at night (ALAN), affects the biodiversity of mammalian and avian species by impairing their cognitive functions. The impact of ALAN on cognition, however, has not been investigated in aquatic species, in spite of the common occurrence of this pollution along water bodies. We exposed eggs of a teleost fish (the zebrafish Danio rerio) to ALAN and, upon hatching, we measured larvae' cognitive abilities with a habituation learning paradigm. Both control and ALAN-exposed larvae showed habituation learning, but the latter learned significantly slower, suggesting that under ALAN conditions, fish require many more events to acquire ecologically relevant information. We also found that individuals' learning performance significantly covaried with two behavioural traits in the control zebrafish, but ALAN disrupted one of these relationships. Additionally, ALAN resulted in an average increase in larval activity. Our results showed that both fish's cognitive abilities and related individual differences are negatively impacted by light pollution, even after a short exposure in the embryonic stage.

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

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

Environmentally relevant concentrations of triclocarban affect behaviour, learning, and brain gene expression in fish.

Many chemicals spilled in aquatic ecosystems can interfere with cognitive abilities and brain functions that control fitness-related behaviour. Hence, their harmful potential may be substantially underestimated. Triclocarban (TCC), one of the most common aquatic contaminants, is known to disrupt hormonal activity, but the consequences of this action on behaviour and its underlying cognitive mechanisms are unclear. We tried to fill this knowledge gap by analysing behaviour, cognitive abilities, and brain gene expression in zebrafish larvae exposed to TCC sublethal concentrations. TCC exposure substantially decreased exploratory behaviour and response to stimulation, while it increased sociability. Additionally, TCC reduced the cognitive performance of zebrafish in a habituation learning task. In the brain of TCC-exposed zebrafish, we found upregulation of c-fos, a gene involved in neural activity, and downregulation of bdnf, a gene that influences behavioural and cognitive traits such as activity, learning, and memory. Overall, our experiments highlight consistent effects of non-lethal TCC concentrations on behaviour, cognitive abilities, and brain functioning in a teleost fish, suggesting critical fitness consequences of these compounds in aquatic ecosystems as well as the potential to affect human health.

Modeling Sarcoglycanopathy in Danio rerio.

Sarcoglycanopathies, also known as limb girdle muscular dystrophy 3-6, are rare muscular dystrophies characterized, although heterogeneous, by high disability, with patients often wheelchair-bound by late adolescence and frequently developing respiratory and cardiac problems. These diseases are currently incurable, emphasizing the importance of effective treatment strategies and the necessity of animal models for drug screening and therapeutic verification. Using the CRISPR/Cas9 genome editing technique, we generated and characterized δ-sarcoglycan and ß-sarcoglycan knockout zebrafish lines, which presented a progressive disease phenotype that worsened from a mild larval stage to distinct myopathic features in adulthood. By subjecting the knockout larvae to a viscous swimming medium, we were able to anticipate disease onset. The δ-SG knockout line was further exploited to demonstrate that a δ-SG missense mutant is a substrate for endoplasmic reticulum-associated degradation (ERAD), indicating premature degradation due to protein folding defects. In conclusion, our study underscores the utility of zebrafish in modeling sarcoglycanopathies through either gene knockout or future knock-in techniques. These novel zebrafish lines will not only enhance our understanding of the disease's pathogenic mechanisms, but will also serve as powerful tools for phenotype-based drug screening, ultimately contributing to the development of a cure for sarcoglycanopathies.

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

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

Adaptive phenotypic plasticity induces individual variability along a cognitive trade-off.

Animal species, including humans, display patterns of individual variability in cognition that are difficult to explain. For instance, some individuals perform well in certain cognitive tasks but show difficulties in others. We experimentally analysed the contribution of cognitive plasticity to such variability. Theory suggests that diametrically opposed cognitive phenotypes increase individuals' fitness in environments with different conditions such as resource predictability. Therefore, if selection has generated plasticity that matches individuals' cognitive phenotypes to the environment, this might produce remarkable cognitive variability. We found that guppies, Poecilia reticulata, exposed to an environment with high resource predictability (i.e. food available at the same time and in the same location) developed enhanced learning abilities. Conversely, guppies exposed to an environment with low resource predictability (i.e. food available at a random time and location) developed enhanced cognitive flexibility and inhibitory control. These cognitive differences align along a trade-off between functions that favour the acquisition of regularities such as learning and functions that adjust behaviour to changing conditions (cognitive flexibility and inhibitory control). Therefore, adaptive cognitive plasticity in response to resource predictability (and potentially similar factors) is a key determinant of cognitive individual differences.

Daily rhythms in the behavioural stress response of the zebrafish Danio rerio.

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.

Sorbed environmental contaminants increase the harmful effects of microplastics in adult zebrafish, Danio rerio.

Aquatic animals ingest Microplastics (MPs) which have the potential to affect the uptake and bioavailability of sorbed co-contaminants. However, the effects on living organisms still need to be properly understood. The present study was designed to assess the combined effects of MPs and environmental contaminants on zebrafish (Danio rerio) health and behavior. Adult specimens were fed according to three different protocols: 1) untreated food (Control group); 2) food supplemented with 0.4 mg/L pristine polyethylene-MPs (PE-MPs; 0.1-0.3 mm diameter) (PEv group); 3) food supplemented with 0.4 mg/L PE-MPs previously incubated (PEi group) for 2 months in seawater. Analysis of contaminants in PEi detected trace elements, such as lead and copper. After 15 days of exposure, zebrafish underwent behavioral analysis and were then dissected to sample gills and intestine for histology, and the latter also for microbiome analysis. Occurrence of PEv and PEi in the intestine and contaminants in the fish carcass were analyzed. Both PEv- and PEi-administered fish differed from controls in the assays performed, but PEi produced more harmful effects in most instances. Overall, MPs after environmental exposure revealed higher potential to alter fish health through combined effects (e.g. proportion of microplastics, pollutants and/or microorganisms).

Early-life exposure to environmentally relevant concentrations of triclocarban impairs ocular development in zebrafish larvae.

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.

bdnf loss affects activity, sociability, and anxiety-like behaviour in zebrafish.

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-/- zebrafish line through a battery of behavioural assays. Results of two behavioural tests suggested higher levels of locomotor activity in bdnf-/- zebrafish compared to bdnf+/+ zebrafish, in spite of similar swimming capacities observed in the two lines in a swim tunnel. bdnf-/- zebrafish also displayed higher levels of attraction towards a social stimulus compared to wild-type zebrafish, suggesting an effect of BDNF on sociability. Last, in a scototaxis test, we found evidence of potential differences in anxiety-like behaviour between the two lines. Our study supports an effect of BDNF on several behavioural traits, in line with early studies in mutant rodent models and in humans, suggesting the possibility to use the zebrafish to investigate diseases that involve alteration in BDNF levels.

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

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

Medaka as a model for seasonal plasticity: Photoperiod-mediated changes in behaviour, cognition, and hormones.

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.

Collagen VI ablation in zebrafish causes neuromuscular defects during developmental and adult stages.

Collagen VI (COL6) is an extracellular matrix protein exerting multiple functions in different tissues. In humans, mutations of COL6 genes cause rare inherited congenital disorders, primarily affecting skeletal muscles and collectively known as COL6-related myopathies, for which no cure is available yet. In order to get insights into the pathogenic mechanisms underlying COL6-related diseases, diverse animal models were produced. However, the roles exerted by COL6 during embryogenesis remain largely unknown. Here, we generated the first zebrafish COL6 knockout line through CRISPR/Cas9 site-specific mutagenesis of the col6a1 gene. Phenotypic characterization during embryonic and larval development revealed that lack of COL6 leads to neuromuscular defects and motor dysfunctions, together with distinctive alterations in the three-dimensional architecture of craniofacial cartilages. These phenotypic features were maintained in adult col6a1 null fish, which displayed defective muscle organization and impaired swimming capabilities. Moreover, col6a1 null fish showed autophagy defects and organelle abnormalities at both embryonic and adult stages, thus recapitulating the main features of patients affected by COL6-related myopathies. Mechanistically, lack of COL6 led to increased BMP signaling, and direct inhibition of BMP activity ameliorated the locomotor activity of col6a1 null embryos. Finally, treatment with salbutamol, a  ß2-adrenergic receptor agonist, elicited a significant amelioration of the neuromuscular and motility defects of col6a1 null fish embryos. Altogether, these findings indicate that this newly generated zebrafish col6a1 null line is a valuable in vivo tool to model COL6-related myopathies and suitable for drug screenings aimed at addressing the quest for effective therapeutic strategies for these disorders.

A thorough annotation of the krill transcriptome offers new insights for the study of physiological processes.

The krill species Euphausia superba plays a critical role in the food chain of the Antarctic ecosystem. Significant changes in climate conditions observed in the Antarctic Peninsula region in the last decades have already altered the distribution of krill and its reproductive dynamics. A deeper understanding of the adaptation capabilities of this species is urgently needed. The availability of a large body of RNA-seq assays allowed us to extend the current knowledge of the krill transcriptome. Our study covered the entire developmental process providing information of central relevance for ecological studies. Here we identified a series of genes involved in different steps of the krill moulting cycle, in the reproductive process and in sexual maturation in accordance with what was already described in previous works. Furthermore, the new transcriptome highlighted the presence of differentially expressed genes previously unknown, playing important roles in cuticle development as well as in energy storage during the krill life cycle. The discovery of new opsin sequences, specifically rhabdomeric opsins, one onychopsin, and one non-visual arthropsin, expands our knowledge of the krill opsin repertoire. We have collected all these results into the KrillDB2 database, a resource combining the latest annotation of the krill transcriptome with a series of analyses targeting genes relevant to krill physiology. KrillDB2 provides in a single resource a comprehensive catalog of krill genes; an atlas of their expression profiles over all RNA-seq datasets publicly available; a study of differential expression across multiple conditions. Finally, it provides initial indications about the expression of microRNA precursors, whose contribution to krill physiology has never been reported before.

Comparison of anxiety-like and social behaviour in medaka and zebrafish.

The medaka, Oryzias latipes, is rapidly growing in importance as a model in behavioural research. However, our knowledge of its behaviour is still incomplete. In this study, we analysed the performance of medaka in 3 tests for anxiety-like behaviour (open-field test, scototaxis test, and diving test) and in 3 sociability tests (shoaling test with live stimuli, octagonal mirror test, and a modified shoaling test with mirror stimulus). The behavioural response of medaka was qualitatively similar to that observed in other teleosts in the open-field test (thigmotaxis), and in 2 sociability tests, the shoaling test and in the octagonal mirror test (attraction towards the social stimulus). In the remaining tests, medaka did not show typical anxiety (i.e., avoidance of light environments and preference for swimming at the bottom of the aquarium) and social responses (attraction towards the social stimulus). As a reference, we compared the behaviour of the medaka to that of a teleost species with well-studied behaviour, the zebrafish, tested under the same conditions. This interspecies comparison indicates several quantitative and qualitative differences across all tests, providing further evidence that the medaka responds differently to the experimental settings compared to other fish models.