The effect of common husbandry conditions (crowding, social environment, water quality, handling, and background color) on the cortisol stress response in adult zebrafish, Danio rerio, was investigated to check the usefulness of zebrafish as a model organism in aquaculture research. In addition, a noninvasive methodology for assessing stress was evaluated. Zebrafish showed a fast cortisol response with high values at 30 min that returned to basal levels within 2 h of poststress. There was a significant positive correlation between trunk cortisol concentrations and the free water cortisol rate (r(2)=0.829-0.850, p<0.001), indicating that measurement of the water-borne cortisol release rate may serve as a noninvasive and reliable stress indicator at the population level. Crowding resulted in 13- to 21-fold greater mean trunk cortisol concentrations compared with controls. However, even at low stocking density (2-5 fish/L), the maintenance cost was higher than the one at higher densities (10 fish/L) due to the formation of dominance hierarchies. The background color affected trunk cortisol concentrations, with fish exposed to brighter backgrounds (green and white) showing 3- to 8-fold greater mean trunk cortisol concentrations than fish exposed to a black background or transparent aquaria. Fish exposed to high stocking densities for 2 h or 5 days had similar high mean trunk cortisol levels, indicating that exposure of fish for the period of 2 h to a specific stressor may represent a chronic situation in zebrafish. It is concluded that adult laboratory zebrafish had a preference for a transparent or black background aquarium, at a number of 10 individuals per 2 L of available water volume, to express their normal behavior and avoid increased cortisol stress reaction.

Zebrafish, Danio rerio, is an emerging model organism in stress and neurobehavioral studies. In nature, the species forms shoals, yet when kept in pairs it exhibits an agonistic and anxiety-like behavior that leads to the establishment of dominant-subordinate relationships. Fluoxetine, a selective serotonin reuptake inhibitor, is used as an anxiolytic tool to alter aggressive behavior in several vertebrates and as an antidepressant drug in humans. Pairs of male zebrafish were held overnight to develop dominant – subordinate behavior, either treated or non-treated for 2 h with fluoxetine (5 mg L-1), and allowed to interact once more for 1 h. Behavior was recorded both prior and after fluoxetine administration. At the end of the experiment, trunk and brain samples were also taken for cortisol determination and mRNA expression studies, respectively. Fluoxetine treatment significantly affected zebrafish behavior and the expression levels of several genes, by decreasing offensive aggression in dominants and by eliminating freezing in the subordinates. There was no statistically significant difference in whole-trunk cortisol concentrations between dominant and subordinate fish, while fluoxetine treatment resulted in higher (P = 0.004) cortisol concentrations in both groups. There were statistically significant differences between dominant and subordinate fish in brain mRNA expression levels of genes involved in stress axis (gr, mr), neural activity (bdnf, c-fos) and the serotonergic system (htr2b, slc6a4b). The significant decrease in the offensive and defensive aggression following fluoxetine treatment was concomitant with a reversed pattern in c-fos expression levels. Overall, an acute administration of a selective serotonin reuptake inhibitor alters aggressive behavior in male zebrafish in association with changes in the neuroendocrine mediators of coping styles.

The Hsd11b2 enzyme converts cortisol into its inactive form, cortisone and regulates cortisol levels, in particular in response to stress. Taking advantage of CRISPR/Cas9 technology, we generated a hsd11b2 zebrafish mutant line to evaluate the involvement of this gene in stress response regulation. The absence of a functional Hsd11b2 affects survival of zebrafish, although homozygous hsd11b2^−/− mutants can reach adulthood. Reproductive capability of hsd11b2^−/− homozygous adult males is almost completely abrogated, while that of females is reduced. Interestingly, basal cortisol levels and glucocorticoid-dependent transcriptional activities are not affected by the mutation. In agreement with basal cortisol results, we also demonstrated that basal response to light (LMR-L/D) or mechanical (VSRA) stimuli is not significantly different in wild-type (hsd11b2^+/+) compared to mutant larvae. However, after exposure to an acute stressor, the cortisol temporal patterns of synthesis and release are prolonged in both 5 days post fertilization larvae and one-year-old adult hsd11b2^−/− zebrafish compared to wild-type siblings, showing at the same time, at 5 dpf, a higher magnitude in the stress response at 10 min post stress. All in all, this new zebrafish model represents a good tool for studying response to different stressors and to identify mechanisms that are induced by cortisol during stress response.

Characteristic manifestations of skin aging, due to either intrinsic or extrinsic factors, such as ultraviolet (UV) radiation and oxidative stress, include cell senescence, alterations in collagen and elastin networks, and melanogenesis disorders. Natural products are considered a rich source of anti-aging molecules. Accordingly, the screening of a plant extract library from the Greek flora for a panel of biological activities related to skin aging is presented herein. In particular, 52 plant materials extracted using Accelerated Solvent Extraction (ASE) and Supercritical Fluid Extraction (SFE) were assessed for their effects on (1) human skin cell viability, (2) antioxidant activity—using both cell-free and cell-based methods—(3) photoprotective capacity, and (4) interference with collagenase, elastase, and tyrosinase, as well as with proteasomal and lysosomal activities of human skin cells. In vivo phenotypic screens on Danio rerio (zebrafish) embryos were also used for assessing melanogenesis. Many active extracts were identified, some of them for the first time, and others in agreement with previous reports. In general, ASE extracts exhibited higher activities than SFE ones. Seven extracts showed multiple activities, being highly effective in at least four different assays. These data support the potential use of these extracts against skin aging in medicinal and cosmetic applications.