Development of autogenous vaccines for farmed European seabass against Aeromonas veronii using zebrafish as a model for efficacy assessment.

Aeromonas veronii bv. sobria is an emerging pathogen for the European seabass cultured in the Aegean Sea (Mediterranean) causing significant problems in the Greek and Turkish aquaculture industry since no licensed vaccine is currently available for the disease. A bivalent vaccine was developed based on two phenotypically distinct strains of the pathogen, PDB (motile, pigment-producing strain) and NS (non-motile, non-pigment-producing). The two strains comprising the bivalent vaccine were evaluated as monovalent products in zebrafish before the seabass trials. Challenges using the homologous or the heterologous strain showed that both vaccines were protective with RPS values ranging between 66 and 100% in zebrafish. The bivalent vaccine was then tested in European seabass following dip or intraperitoneal administration. Efficacy was evaluated separately against both strains comprising the bivalent vaccine. Dip vaccination applied to juvenile seabass of 2.5 g average weight provided protection following challenge tests 30 days post vaccination only in one of the two strains tested (strain PDB, RPS: 88%). This was also the case in the injection vaccination of adult seabass of 60 g average weight where the vaccine was effective only against the PDB strain (RPS: 63%). High antibody titers against both strains were found at 30 and 60 days after intraperitoneal vaccination in the adult seabass. The use of zebrafish as a model for vaccine development for aquaculture species is discussed.

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.

Water temperature modifies the acute stress response of European sea bass, Dicentrarchus labrax L. (1758).

The effects of water temperature (15, 20 and 25 °C) on the stress response of European sea bass, Dicentrarchus labrax, were studied. Blood and water samples were collected prior and at 0.5, 1, 2, 4 and 8 h post-stress for hormonal and biochemical analysis. Water temperature affected the resting concentrations of all stress indicators examined, as well as their response after stress, apart from lactate which response was unaffected by temperature. Cortisol showed the response with the highest outcome at 15 °C, and the fastest at 25 °C. Cortisol release rate in the water was also faster and of higher magnitude at higher temperatures. Glucose had both the fastest and higher in magnitude response at 25 °C, while lactate responded similarly in all temperatures tested. Finally, osmolality changed only at the highest temperature. These results suggest that temperature should be taken into consideration when evaluating the resting levels of stress indicators of European sea bass in laboratory, rearing and field conditions. Additionally, the present results show that temperature affects the stress response, suggesting that fish can differ in their susceptibility to stress depending on the acclimatization temperature. Moreover, it was shown that cortisol release rate in the water, a non-invasive stress indicator, can depict the effects of temperature on cortisol stress response.

Refined analytical pipeline for the pharmacodynamic assessment of T-cell responses to vaccine antigens.

Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.

Programmed Intermittent Epidural Boluses of 0.1% Ropivacaine Versus 0.2% Ropivacaine for the Maintenance of Epidural Analgesia in Labor.

Objective The gold standard for pain management during labor is epidural analgesia, which can be administered in two different ways to the parturients, either by bolus doses or continuous infusions of local anesthetic solutions with opioids. Recently, programmed intermittent epidural boluses (PIEBs) via a pump are gaining popularity as a very effective method with minimal side effects. The aim of this study was to evaluate the optimum ropivacaine concentration between two different regimens (0.1% or 0.2% both with fentanyl 2 µg/ml) that can provide satisfactory analgesia with the minimum degree of motor blockade, using PIEBs. Methods A prospective randomized controlled study was performed from March 2020 to March 2022. Two different concentrations of ropivacaine 0.1% and 0.2% via PIEBs were equally allocated to two groups of parturients with an additional patient control epidural rescue bolus if needed. Our primary endpoint was motor blockade, as assessed by the modified Bromage scale (MBS). We also recorded visual analog scale (VAS) scores, heart rate, blood pressure, total local anesthetic consumption, labor duration and method of delivery, and APGAR score of the newborns. Results All patients presented Bromage scores equal to 6, and the total consumption of the anesthetic solution was comparable between the two groups. Women in the 0.2% group showed higher pain relief and satisfaction compared to the 0.1% group. Concerning the 0,2% group, diastolic blood pressure and APGAR scores were lower alongside with a lower satisfactory extrusion stage observed by the obstetrician. Conclusion Both ropivacaine regimens provide satisfactory labor epidural analgesia for the expectant mother without any motor blockade.

Neuronal atg1 Coordinates Autophagy Induction and Physiological Adaptations to Balance mTORC1 Signalling.

The mTORC1 nutrient-sensing pathway integrates metabolic and endocrine signals into the brain to evoke physiological responses to food deprivation, such as autophagy. Nevertheless, the impact of neuronal mTORC1 activity on neuronal circuits and organismal metabolism remains obscure. Here, we show that mTORC1 inhibition acutely perturbs serotonergic neurotransmission via proteostatic alterations evoked by the autophagy inducer atg1. Neuronal ATG1 alters the intracellular localization of the serotonin transporter, which increases the extracellular serotonin and stimulates the 5HTR7 postsynaptic receptor. 5HTR7 enhances food-searching behaviour and ecdysone-induced catabolism in Drosophila. Along similar lines, the pharmacological inhibition of mTORC1 in zebrafish also stimulates food-searching behaviour via serotonergic activity. These effects occur in parallel with neuronal autophagy induction, irrespective of the autophagic activity and the protein synthesis reduction. In addition, ectopic neuronal atg1 expression enhances catabolism via insulin pathway downregulation, impedes peptidergic secretion, and activates non-cell autonomous cAMP/PKA. The above exert diverse systemic effects on organismal metabolism, development, melanisation, and longevity. We conclude that neuronal atg1 aligns neuronal autophagy induction with distinct physiological modulations, to orchestrate a coordinated physiological response against reduced mTORC1 activity.

DNA based neoepitope vaccination induces tumor control in syngeneic mouse models.

Recent findings have positioned tumor mutation-derived neoepitopes as attractive targets for cancer immunotherapy. Cancer vaccines that deliver neoepitopes via various vaccine formulations have demonstrated promising preliminary results in patients and animal models. In the presented work, we assessed the ability of plasmid DNA to confer neoepitope immunogenicity and anti-tumor effect in two murine syngeneic cancer models. We demonstrated that neoepitope DNA vaccination led to anti-tumor immunity in the CT26 and B16F10 tumor models, with the long-lasting presence of neoepitope-specific T-cell responses in blood, spleen, and tumors after immunization. We further observed that engagement of both the CD4+ and CD8+ T cell compartments was essential to hamper tumor growth. Additionally, combination therapy with immune checkpoint inhibition provided an additive effect, superior to either monotherapy. DNA vaccination offers a versatile platform that allows the encoding of multiple neoepitopes in a single formulation and is thus a feasible strategy for personalized immunotherapy via neoepitope vaccination.

Fish Scales Produce Cortisol upon Stimulation with ACTH.

Cortisol concentration in fish scales is a novel and reliable indicator of chronic stress. However, until now cortisol in scales has been considered to be accumulated through the circulation and it has not yet been studied whether it can be de novo produced from cells found in the scales. In the current study, scales of European sea bass, Dicentrarchus labrax, were stimulated in-vitro with a range of concentrations of adrenocorticotropic hormone (ACTH) to investigate if they can produce and release cortisol. Moreover, scales were exposed to a combination of ACTH and metyrapone, an inhibitor of cortisol production, to examine whether cortisol was actually produced in the scales. Results from ACTH administration showed that scales increased their cortisol release in a dose-dependent manner. This effect was reversed when scales were co-incubated with ACTH and metyrapone, indicating that cortisol was produced de novo and not released only upon stimulation with ACTH.

Metabolic scope, performance and tolerance of juvenile European sea bass Dicentrarchus labrax upon acclimation to high temperatures.

European sea bass is a species of great commercial value for fisheries and aquaculture. Rising temperatures may jeopardize the performance and survival of the species across its distribution and farming range, making the investigation of its thermal responses highly relevant. In this article, the metabolic scope, performance, and tolerance of juvenile E. sea bass reared under three high water temperatures (24, 28, 33°C), for a period of three months was evaluated via analysis of selected growth performance and physiological indicators. Effects on molecular, hormonal, and biochemical variables were analyzed along with effects of acclimation temperature on the metabolic rate and Critical Thermal maximum (CTmax). Despite signs of thermal stress at 28°C indicated by high plasma cortisol and lactate levels as well as the upregulation of genes coding for Heat Shock Proteins (HSP), E. sea bass can maintain high performance at that temperature which is encouraging for the species culture in the context of a warming ocean. Critical survivability thresholds appear sharply close to 33°C, where the aerobic capacity declines and the overall performance diminishes. European sea bass demonstrates appreciable capacity to cope with acute thermal stress exhibiting CTmax as high as 40°C for fish acclimated at high temperatures, which may indicate resilience to future heatwaves events.

Genomic Selection and Genome-Wide Association Analysis for Stress Response, Disease Resistance and Body Weight in European Seabass.

The majority of the genetic studies in aquaculture breeding programs focus on commercial traits such as body weight, morphology, and resistance against diseases. However, studying stress response in European seabass may contribute to the understanding of the genetic component of stress and its future use to select broodstock whose offspring may potentially be less affected by handling. A total of 865 European seabass offspring were used to measure body weight and stress response. Moreover, a disease challenge experiment with Vibrio anguillarum was conducted in a subset (332) of the above fish to study disease resistance. Fish were genotyped with a 57k SNP array, and a Genome-Wide Association study (GWAS) was performed. Five SNPs were found to be statistically significant, three of which affect stress indicators and body weight (in a subgroup of the population), and a putative SNP affects growth performance, while no SNP associated with resistance to Vibrio was found. A moderate to high genomic heritability regarding stress indicators and body weight was estimated using the Restricted Maximum Likelihood (REML) process. Finally, the accuracy, along with the correlation between Estimated Breeding Values (EBVs) and Genomic Estimated Breeding Values (GEBVs), were calculated for all the traits.

A Modified Protocol for Cortisol Analysis in Zebrafish (Danio rerio), Individual Embryos, and Larvae.

A modified protocol for the extraction and analysis of cortisol in individual zebrafish, Danio rerio, embryo, and larva samples has been developed and evaluated. Recovery efficiency of the method was high, specifically calculated at 93.8% ± 6.5%. Dilution tests showed high parallelism, while increasing the number of individuals used in each extraction sample resulted in a linear, although slightly underestimated, increase of cortisol yield. Results of cortisol content from 0, 3, and 5 days postfertilization (dpf) fish using the proposed protocol were within the range of most published studies analyzing cortisol in pooled samples of 10-30 individuals. Moreover, 5 dpf larvae had significantly higher cortisol levels than embryos, a pattern commonly observed in literature. Finally, application of an osmotic stress in 5 dpf larvae led to a statistically significant increase in cortisol content.

Behavioural and physiological responses to a conditioning protocol for adult zebrafish, Danio rerio, held in groups.

The current study developed and evaluated a conditioning protocol for zebrafish which can be used in groups of fish instead of isolated individuals. The protocol consisted of the application of a neutral conditioned stimulus (CS), i.e. a novel object in the tank, followed by an aversive, stressful unconditioned stimulus (US), i.e. chasing, in groups of fish in experimental tanks. This training protocol was applied to fish once per day for one- or three- consecutive days. The following day fish were tested for the display of behavioural, i.e. bottom dwelling, and neuroendocrine, i.e. cortisol and stress-related gene expression, conditioned responses. Results showed that one-day of conditioning was not adequate to produce conditioned responses. Repetition of training sessions for three days, however, led to the display of conditioned responses upon exposure to the conditioning stimulus alone. Specifically, when exposed to the CS, paired fish showed increased bottom dwelling, as well as elevated trunk cortisol concentration and c-fos gene expression in the brain. Data suggest that the applied CS can be perceived by fish as a signal to prepare anticipatory behavioural and neuroendocrine responses to cope with the expected aversive event. Finally, this study is one of the few to test associative learning in groups of zebrafish, thus avoiding training and testing fish individually which can be stressful and impede normal behaviour.

QTL for Stress and Disease Resistance in European Sea Bass, Dicentrarhus labrax L.

There is a growing interest in selective breeding in European sea bass (Dicentrarchus labrax), especially regarding family selection based on growth performance. In particular, quantitative trait loci (QTL) identification in sea bass enhances the application of marker-assisted breeding for the genetic improvement of the production traits. The aims of the study were to identify potential QTL affecting stress and immunological indicators, body weight, and mortality after vibriosis injection in sea bass as well as to estimate heritability and genetic/phenotypic correlations for the aforementioned traits. To this end, stress test was performed on 960 offspring and a sub-group of them (420) was selected to explore the mortality after vibrio injection. Selective genotyping was performed in 620 offspring for 35 microsatellite markers and distributed into 6 linkage groups. The length of the genetic linkage map was 283.6 cM and the mean distance between the markers was 8.1 cM. QTL affecting body weight in three different growth periods detected on linkage groups LG1, LG4, LG6, and LG14. A QTL associated with weight in early growth stages (290-306 days post-hatching) was also identified on LG3. QTL analysis confirmed the existence of QTL affecting cortisol levels, on LG3 and LG14. Moreover, new QTL affecting only cortisol and glucose levels were detected on LG1 and LG23. No QTL affecting hormonal or biochemical marks was found on LG4 and LG6. Heritability of cortisol, lysozyme levels, and mortality were high (0.36, 0.55, and 0.38, respectively).

Characterization of a Highly Virulent Edwardsiella anguillarum Strain Isolated From Greek Aquaculture, and a Spontaneously Induced Prophage Therein.

Edwardsiella-associated outbreaks are increasingly reported on both marine and freshwater aquaculture setups, accounting for severe financial and biomass losses. E. tarda, E. ictaluri, and E. hoshinae have been the traditional causative agents of edwardsiellosis in aquaculture, however, intensive studies due to the significance of the disease have just recently revealed two more species, E. piscicida and E. anguillarum. Whole genome sequencing that was conducted on the strain EA011113, isolated from farmed Diplodus puntazzo after an edwardsiellosis outbreak in Greece, confirmed it as a new clinical strain of E. anguillarum. Extensive phylogenetic analysis showed that this Greek strain is closely related to an Israeli E. piscicida-like clinical strain, isolated from diseased groupers, Epinephelus aeneus and E. marginatus in Red Sea. Bioinformatic analyses of E. anguillarum strain EA011113 unveiled a wide repertoire of potential virulence factors, the effect of which was corroborated by the mortalities that the strain induced in adult zebrafish, Danio rerio, under different levels of infection intensity (LD50 after 48 h: 1.85 × 104 cfu/fish). This strain was non-motile and according to electron microscopy lacked flagella, a fact that is not typical for E. anguillarum. Comparative genomic analysis revealed a deletion of 36 nt found in the flagellar biosynthetic gene (FlhB) that could explain that trait. Further in silico analysis revealed an intact prophage that was integrated in the bacterial genome. Following spontaneous induction, the phage was isolated, purified, characterized and independently sequenced, confirming its viability as a free, inducible virion as well. Separate genomic analysis of the prophage implies a plausible case of lysogenic conversion. Focusing on edwardsiellosis as a rapidly emerging aquaculture disease on a global scale, this work offers some insight into the virulence, fitness, and potential lysogenic conversion of a of a newly described, yet highly pathogenic, strain of E. anguillarum.

Regulation of divergent cortisol responsiveness in European sea bass, Dicentrarchus labrax L.

Mechanisms regulating differences in cortisol responsiveness between low (LR) and high response (HR) individuals have been poorly studied. In this context, we aimed to study key regulatory processes in cortisol dynamics at the head kidneys of LR and HR European sea bass. To do so, resting plasma cortisol and ACTH concentrations were quantified in these fish. Additionally, the head kidneys of these individuals were superfused through an in vitro superfusion system and stimulated with the same amount of ACTH to assess their cortisol biosynthetic capacity. Moreover, the expression of important genes in cortisol regulation was assessed. Results showed that LR fish had lower resting cortisol concentrations than HR, although no differences existed in the circulating levels of ACTH. Additionally, the biosynthetic capacity of HR was higher than that of LR fish when in vitro stimulated with ACTH. At the molecular level, a statistically significant 3.4-fold higher expression of the ACTH receptor, mc2r, and a 2.3-fold, though not significant, higher expression of 11ß-hydroxylase (cyp11b1), an enzyme involved in cortisol biosynthesis, was observed in the HR fish. Finally, a statistically significant 1.3-fold lower expression of 11ß-hydroxysteroid dehydrogenase 2 (hsd11b2), an enzyme involved in cortisol inactivation, was observed in HR when compared to LR fish. Therefore, it was for the first time indicated that cortisol dynamics can also be regulated at the post-production level in the head kidney. Collectively, our results highlight the crucial role of the interrenal tissue in the regulation of differences in cortisol response between LR and HR sea bass individuals.

Early life stress induces long-term changes in limbic areas of a teleost fish: the role of catecholamine systems in stress coping.

Early life stress (ELS) shapes the way individuals cope with future situations. Animals use cognitive flexibility to cope with their ever-changing environment and this is mainly processed in forebrain areas. We investigated the performance of juvenile gilthead seabream, previously subjected to an ELS regime. ELS fish showed overall higher brain catecholaminergic (CA) signalling and lower brain derived neurotrophic factor (bdnf) and higher cfos expression in region-specific areas. All fish showed a normal cortisol and serotonergic response to acute stress. Brain dopaminergic activity and the expression of the α2Α adrenergic receptor were overall higher in the fish homologue to the lateral septum (Vv), suggesting that the Vv is important in CA system regulation. Interestingly, ELS prevented post-acute stress downregulation of the α2Α receptor in the amygdala homologue (Dm3). There was a lack of post-stress response in the ß2 adrenergic receptor expression and a downregulation in bdnf in the Dm3 of ELS fish, which together indicate an allostatic overload in their stress coping ability. ELS fish showed higher neuronal activity (cfos) post-acute stress in the hippocampus homologue (Dlv) and the Dm3. Our results show clear long-term effects on limbic systems of seabream that may compromise their future coping ability to environmental challenges.

Allostatic Load and Stress Physiology in European Seabass (Dicentrarchus labrax L.) and Gilthead Seabream (Sparus aurata L.).

The present study aimed to compare effects of increasing chronic stress load on the stress response of European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) to identify neuroendocrine functions that regulate this response. Fish were left undisturbed (controls) or exposed to three levels of chronic stress for 3 weeks and then subjected to an acute stress test (ACT). Chronic stress impeded growth and decreased feed consumption in seabass, not in seabream. In seabass basal cortisol levels are high and increase with stress load; the response to a subsequent ACT decreases with increasing (earlier) load. Basal cortisol levels in seabream increase with the stress load, whereas the ACT induced a similar response in all groups. In seabass and seabream plasma α-MSH levels and brain stem serotonergic activity and turnover were similar and not affected by chronic stress. Species-specific molecular neuro-regional differences were seen. In-situ hybridization analysis of the early immediate gene cfos in the preoptic area showed ACT-activation in seabream; in seabass the expression level was not affected by ACT and seems constitutively high. In seabream, expression levels of telencephalic crf, crfbp, gr1, and mr were downregulated; the seabass hypothalamic preoptic area showed increased expression of crf and gr1, and decreased expression of mr, and this increased the gr1/mr ratio considerably. We substantiate species-specific physiological differences to stress coping between seabream and seabass at an endocrine and neuroendocrine molecular level. Seabass appear less resilient to stress, which we conclude from high basal activities of stress-related parameters and poor, or absent, responses to ACT. This comparative study reveals important aquaculture, husbandry, and welfare implications for the rearing of these species.

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.

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.

Husbandry of zebrafish, Danio rerio, and the cortisol stress response.

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.