Knockout of the hsd11b2 Gene Extends the Cortisol Stress Response in Both Zebrafish Larvae and Adults.

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.

Thermal tolerance, metabolic scope and performance of meagre, Argyrosomus regius, reared under high water temperatures.

This article reports on the thermal tolerance, metabolic capacity and performance of juvenile meagre (Argyrosomus regius) reared under three high water temperatures (24, 29 and 34 °C) for three months. The analysis includes the thermal effects on the growth performance, metabolism and physiology of meagre, including a range of molecular, haematological, metabolic, enzymatic and hormonal indicators, as well as the effects on the proximate composition and ingestion speed. Meagre performs best between 24 and 29 °C while the temperature of 34 °C is very close to the upper end of its temperature tolerance range. At 34 °C meagre exhibits a poor growth performance and physiological status, increased blood clotting, high mortality rates and a diminished capacity for aerobic metabolism, as indicated by its low aerobic scope (129 mg kg-1 h-1). Meagre may tolerate short exposures to high temperatures after sufficient acclimation (Critical thermal maximum of 37.5 °C after acclimation to 29 °C) but its overall performance declines under prolonged exposure, suggesting that this emerging aquaculture species may be vulnerable to global warming. Our work corroborates previous findings on the thermal preferences of the species, identifies critical biological thresholds, and provides insights into the effects of prolonged exposure to high temperature regimes.

Inventory of European Sea Bass (Dicentrarchus labrax) sncRNAs Vital During Early Teleost Development.

During early animal ontogenesis, a plethora of small non-coding RNAs (sncRNAs) are greatly expressed and have been shown to be involved in several regulatory pathways vital to proper development. The rapid advancements in sequencing and computing methodologies in the last decade have paved the way for the production of sequencing data in a broad range of organisms, including teleost species. Consequently, this has led to the discovery of sncRNAs as well as the potentially novel roles of sncRNA in gene regulation. Among the several classes of sncRNAs, microRNAs (miRNAs) have, in particular, been shown to play a key role in development. The present work aims to identify the miRNAs that play important roles during early European sea bass (Dicentrarchus labrax) development. The European sea bass is a species of high commercial impact in European and especially Mediterranean aquaculture. This study reports, for the first time, the identification and characterization of small RNAs that play a part in the 10 developmental stages (from morula to all fins) of the European sea bass. From 10 developmental stages, more than 135 million reads, generated by next-generation sequencing, were retrieved from publicly available databases as well as newly generated. The analysis resulted in about 2,000 sample grouped reads, and their subsequently annotation revealed that the majority of transcripts belonged to the class of miRNAs followed by small nuclear RNAs and small nucleolar RNAs. The analysis of small RNA expression among the developmental stages under study revealed that miRNAs are active throughout development, with the main activity occurring after the earlier stages (morula and 50% epiboly) and at the later stages (first feeding, flexion, and all fins). Furthermore, investigating miRNAs exclusively expressed in one of the stages unraveled five miRNAs with a higher abundance only in the morula stage (miR-155, miR-430a, d1, d2, and miR-458), indicating possible important key roles of those miRNAs in further embryonic development. An additional target search showed putative miRNA-mRNA interactions with possible direct and indirect regulatory functions of the identified miRNAs.

Acute Exposure to Fluoxetine Alters Aggressive Behavior of Zebrafish and Expression of Genes Involved in Serotonergic System Regulation.

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.

Unusual α-Carbon Hydroxylation of Proline Promotes Active-Site Maturation.

The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexplored in bacteria. We describe here a so far unknown Pro hydroxylation activity which occurs in active sites of polysaccharide deacetylases (PDAs) from bacterial pathogens, modifying the protein backbone at the Cα atom of a Pro residue to produce 2-hydroxyproline (2-Hyp). This process modifies with high specificity a conserved Pro, shares with the deacetylation reaction the same active site and one catalytic residue, and utilizes molecular oxygen as source for the hydroxyl group oxygen of 2-Hyp. By providing additional hydrogen-bonding capacity, the Pro→2-Hyp conversion alters the active site and enhances significantly deacetylase activity, probably by creating a more favorable environment for transition-state stabilization. Our results classify this process as an active-site "maturation", which is highly atypical in being a protein backbone-modifying activity, rather than a side-chain-modifying one.

Neuroendocrine regulation of the stress response in adult zebrafish, Danio rerio.

The main objectives of this study were to investigate the dynamics of the cortisol stress response and the underlying molecular regulation in adult zebrafish exposed to acute and long-term stressors that differed in nature, duration and relative intensity. Fish showed a very rapid and prolonged increase in trunk cortisol concentrations, starting at around 15min and returning to basal levels at around 2h following exposure to acute stressors. In addition, acute stress affected significantly brain mRNA expression levels of several genes (corticotropin-releasing factor, crf; pro-opiomelanocortin, pomc; glucocorticoid receptor, gr; MR/GR ratio; prolactin, prl; hypocretin/orexin, hcrt; brain-derived neurotrophic factor, bdnf; c-fos). Exposure of fish to unpredictable relatively low-grade environmental and husbandry stressors (SP-1) did not affect the overall behaviour of fish, as well as trunk cortisol concentrations. Fish exposed to relatively higher-grade long-term stressors (SP-2) showed elevated cortisol levels as well as significant changes in most of gene transcripts. In particular, fish exposed to SP-2 showed statistically significant upregulation in brain gr, mr, prl and hcrt compared to SP-1 and control individuals. The highest mean values of bdnf transcripts were found in SP-2 exposed zebrafish and the lowest in control fish, while an approximately 5 to 6-fold upregulation was observed in c-fos mean relative mRNA levels of long-term stress-exposed fish, regardless of stressor intensity, compared to control zebrafish. In conclusion, we developed realistic acute and unpredictable long-term stress protocols, based on husbandry and environmental stressors and physical, chemical, mechanical and social stimuli that fish may experience either in nature or under intensive rearing conditions.

Purification, crystallization and preliminary X-ray analysis of the peptidoglycan N-acetylglucosamine deacetylase BC1960 from Bacillus cereus in the presence of its substrate (GlcNAc)6.

The peptidoglycan N-acetylglucosamine (GlcNAc) deacetylase BC1960 from Bacillus cereus (EC 3.5.1.33), an enzyme consisting of 275 amino acids, was crystallized in the presence of its substrate (GlcNAc)(6). The crystals belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 92.7, c = 242.9 A and four molecules in the asymmetric unit. A complete data set was collected at 100 K to a resolution of 2.38 A using synchrotron radiation.