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.

Environmental enrichment decreases anxiety-like behavior in zebrafish larvae.

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

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.

The Application of Synthetic Flavors in Zebrafish (Danio rerio) Rearing with Emphasis on Attractive Ones: Effects on Fish Development, Welfare, and Appetite.

The aim of the present study was to test synthetic flavors as potential feed attractants in zebrafish (Danio rerio) during early development. Six experimental groups were set up in triplicate: (i) a CTRL group fed a zebrafish commercial diet; (ii) a PG group fed a control diet added with Propylene Glycol (PG); (iii) A1+ and A2+ groups fed a control diet added with 1% of the two attractive flavors (A1+ cheese odor made by mixing Propylene Glycol (PG) with the aromatic chemicals trimethyamine, 2-acetylpyrazine, 2-acetylpyridine, and dimethyl sulfide; and A2+ caramel odor, made of PG mixed with the aromatic chemicals vanillin, maltol, cyclotene, acetoin, butyric acid, and capric acid with traces of both gamma-octalactone and gamma-esalactone) or the repulsive flavor (A- coconut odor, made by mixing PG with the aromatic chemicals gamma-eptalactone, gamma-nonalactone, delta-esalactone, and vanillin with trace of both delta-octalactone and maltol), respectively; (iv) an ROT group fed the two attractive diets, each administered singularly in a weekly rotation scheme. All the tested synthetic flavors did not affect the overall health of larval and juvenile fish and promoted growth. Due to the longer exposure time, results obtained from the juvenile stage provided a clearer picture of the fish responses: zebrafish fed both attractive diets showed higher appetite stimulus, feed ingestion, and growth, while the brain dopaminergic activity suggested the A2+ diet as the most valuable solution for its long-lasting effect over the whole experiment (60-day feeding trial, from larvae to adults). The present study provided important results about the possible use of attractive synthetic flavors for aquafeed production, opening new sustainable and more economically valuable opportunities for the aquaculture sector.

A novel-odour exploration test for measuring anxiety in adult and larval zebrafish.

BACKGROUND: Existing methods to assess anxiety in zebrafish are mostly based on visual exploration. However, evidence suggests that zebrafish are more attuned to olfactory than visual stimuli. We developed a novel-odour exploration test (NOEt) for zebrafish. NEW METHOD: Adult zebrafish were exposed to a stimulus sponge soaked with olfactory cue and a control sponge with no cue, placed at the extremities of a narrow, rectangular tank. We scored time spent close to the two sponges to calculate the attraction towards the olfactory cue. In experiment 1, we tested adult zebrafish in the NOEt using various olfactory cues. In experiment 2, we tested larvae. In experiment 3, we compared the NOEt with the most used method to assess anxiety, the open-field test. RESULTS: In experiment 1, zebrafish responded markedly to cues, by either approaching or avoiding the soaked sponge according to the type of cue. The temporal pattern of exploration toward the cue was similar to that of novel objects' exploration in this species. In experiment 2, larvae responded to novel odours, but differently from adults. In experiment 3, we found a relationship between the NOEt and the open-field test. COMPARISON WITH EXISTING METHOD(S): Compared to existing tests, the NOEt has the advantage of exploiting the preferred sensory modality of zebrafish. Moreover, it can be used in early stages because olfactory receptors develop early in this species. CONCLUSIONS: The NOEt is a simple, rapid and low-cost test to study anxiety in zebrafish using the spontaneous exploration of novel olfactory cues.