Michail Pavlidis

Species specificity in the magnitude and duration of the acute stress response in Mediterranean marine fish in culture.

The aim of the present study was to examine the species-specific stress response for seven Mediterranean fishes in culture. Also, to evaluate the method of measuring free cortisol concentration in the rearing water as a non-invasive and reliable indicator of stress in marine species, of aquaculture importance. Gilthead sea bream, Sparus aurata (Sparidae); common dentex, Dentex dentex (Sparidae); common Pandora, Pagellus erythrinus (Sparidae); sharpsnout sea bream, Diplodus puntazzo (Sparidae); dusky grouper, Epinephelus marginatus (Serranidae); meagre, Argyrosomus regius (Sciaenidae) and European sea bass, Dicentrarchus labrax (Moronidae) were subjected to identical acute stress (5-6 min chasing and 1-1.5 min air exposure) under the same environmental conditions and samples were analyzed by the same procedures. Results indicated that there was a clear species-specificity in the magnitude, timing and duration of the stress response in terms of cortisol, glucose and lactate. European sea bass showed a very high response and dusky grouper and meagre a very low response, except plasma glucose concentrations of dusky grouper which was constantly high, while sharpsnout sea bream presented a protracted stress response, up to 8h. The present study confirmed that free cortisol release rate into the water can be used as a reliable stress indicator.

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.

Evaluation of transportation procedures on water quality and fry performance in red porgy (Pagrus pagrus) fry

Abstract The effect of stocking density (10, 20, 30, 40 kg/m3), water renewal (0% or 100% renewal rate per hour), hauling temperature (14, 19, 24 °C), salinity (20, 25, 30, 35, 40 psu), and use of anaesthesia (0, 10, 20, or 50 ppm ethynelglycol-monophenylether) prior to transportation on red porgys fry performance, liver glycogen, and water quality parameters was tested to evaluate transportation procedures in a promising candidate for aquaculture. Simulated transport was performed in plastic containers (volume 60 l) for 48 h. Water samples were taken at 4-h intervals after transport for the determination of pH, carbon dioxide (CO2), un-ionised ammonia (NH3) and ammonium (NH4+). Additionally, liver samples were collected at 4 and 48 h for glycogen determination. There were no statistically significant fluctuations in dissolved CO2 concentration in all tested conditions. Stocking density did not affect NH3 and NH4+ average values and hepatic glycogen content in groups exposed to a water renewal rate of 100%, while increasing NH3 and NH4+ average values with increasing stocking density was observed in groups with no water renewal. Under the same stocking density, a significant change in NH3 and NH4+ fluctuations over the duration of the experiment was observed with concentrations increasing, with a mean exponential rate (±SD) of 0.060±0.005 (NH3) and 0.062±0.005 (NH4+) per hour in groups with no water renewal, and −0.033±0.004 (NH3) and −0.024±0.007 (NH4+) per hour in groups with 100% water renewal. Water temperature affected significantly the hepatic glycogen content and survival during transport. There was no significant effect of salinity and anaesthetic (except at a dose of 50 ppm) on fry survival and on the water quality parameters. It is suggested that red porgy should be transported in stocking densities of 20–25 kg/m3 and at a hauling temperature similar to that kept at the exporters fish rearing tanks (preferable 19 °C). Besides, it is recommended to avoid temperature differences between the hauling water and the water used for renewal during fry transportation.

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.

Ontogenesis of the HPI axis and molecular regulation of the cortisol stress response during early development in Dicentrarchus labrax .

The cortisol stress response and the molecular programming of the corticoid axis were characterized for the first time during early ontogeny in a Mediterranean marine teleost, the European sea bass (Dicentrarchus labrax). Sea bass embryos, pre-larvae and larvae at specific points of development were exposed to acute stressors and the temporal patterns of cortisol whole body concentrations and the expression of genes involved in corticosteroid biosynthesis, degradation and signaling were determined. Expression of genes (gr1, gr2, mr, crf) involved into the corticoid response regulation combined with histological data indicated that, although a cortisol stress response is evident for the first time around first feeding, a pattern becomes established in larvae at flexion until the formation of all fins. Moreover, mRNA transcript levels of 11β-hydroxylase and 11β-hsd2 showed a strong correlation with the whole body cortisol concentrations. Concluding, our data reveal the presence of an adaptive mechanism in European sea bass at early ontogeny enabling to cope with external stressful stimuli and provide a better insight into the onset and regulation of the stress response in this 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.

Early life low intensity stress experience modifies acute stress effects on juvenile brain cell proliferation of European sea bass (D. Labrax)

HighlightsJuvenile sea bass brain is characterized by extensive active cell proliferation.Early life adversity (UCLIS) did not change juvenile brain cell proliferation.Acute stress reduced mitotic activity in teleost hippocampus, amygdala, cerebellum.Acute stress increased plasma cortisol in all cases (non‐UCLIS, UCLIS juveniles).Early UCLIS modified acute stress plasticity of brain cell proliferation responses. ABSTRACT Early life adversity may be critical for the brain structural plasticity that in turn would influence juvenile behaviour. To address this, we questioned whether early life environment has an impact on stress responses latter in life, using European sea bass, Dicentrarchus labrax, as a model organism. Unpredictable chronic low intensity stress (UCLIS), using a variety of moderate intensity stressors, was applied during two early ontogenetic stages, flexion or formation all fins. At juvenile stage, fish were exposed to acute stress and plasma cortisol, brain mRNA expression of corticosteroid receptors’ genes (gr1, gr2, mr) and brain cell proliferation (using BrdU immunohistochemistry) were determined in experimental and matched controls. UCLIS treatment specifically decreased brain gr1 expression in juveniles, but had no effect on the juvenile brain cell proliferation pattern within the major neurogenic zones studied of dorsal (Dm, Dld) and ventral (Vv) telencephalic, preoptic (NPO) areas, periventricular tectum gray zone (PGZ) and valvula cerebellum (VCe). In contrast, exposure to acute stress induced significant plasma cortisol rise, decreases of cerebral cell proliferation in juveniles, not previously exposed to UCLIS, but no effect detected on the expression levels of gr1, gr2 and mr in all groups of different early life history. Interestingly, juveniles with UCLIS history showed modified responses to acute stress, attenuating acute stress‐induced cell proliferation decreases, indicating a long‐lasting effect of early life treatment. Taken together, early life mild stress experience influences an acute stress plasticity end‐point, cerebral cell proliferation, independently of the stress‐axis activation, possibly leading to more effective coping styles.

Repeatability of cortisol stress response in the European sea bass (Dicentrarchus labrax) and transcription differences between individuals with divergent responses

Understanding the stress responses of organisms is of importance in the performance and welfare of farmed animals, including fish. Especially fish in aquaculture commonly face stressors, and better knowledge of their responses may assist in proper husbandry and selection of breeding stocks. European sea bass (Dicentrarchus labrax), a species with high cortisol concentrations, is of major importance in this respect. The main objectives of the present study were to assess the repeatability and consistency of cortisol stress response and to identify differences in liver transcription profiles of European sea bass individuals, showing a consistent low (LR) or high (HR) cortisol response. The progeny of six full sib families was used, and sampled for plasma cortisol after an acute stress challenge once per month, for four consecutive months. Results suggest that cortisol responsiveness was a repeatable trait with LR and HR fish showing low or high resting, free and post-stress cortisol concentrations respectively. Finally, the liver transcription profiles of LR and HR fish showed some important differences, indicating differential hepatic regulation between these divergent phenotypes. These transcription differences were related to various metabolic and immunological processes, with 169 transcripts being transcribed exclusively in LR fish and 161 exclusively in HR fish.

Do experimental units of different scale affect the biological performance of European sea bass Dicentrarchus labrax larvae

The effects of different tank volumes (2000, 500 and 40 l) on European sea bass Dicentrarchus labrax larval rearing, relating to growth, survival, quality and stress variables, were investigated. A dynamic energy budget (DEB) model was used to analyse the results. The hydrodynamics of the tanks exhibited differences, with the water currents in the 2000 l tanks to be almost one order of magnitude stronger than those in the 40 l ones. Important differences in fish growth were observed between small and large tank-rearing volumes, with the smallest tank resulting in the slowest growth. Based on the DEB model analysis, growth differences were related to feeding rates, with growth in the smaller tank limited by food availability. Differences in survival rates were not statistically significant among the tank-rearing volumes. The quality evaluation of the fry (in terms of swimbladder, jaw and skeletal abnormalities) showed differences, with the smallest tank having the highest percentage of deformed individuals. This could be attributed to both the feeding variances and the hydrodynamics in the tanks. No differences were observed in terms of whole-body cortisol at the two developmental stages; flexion, and when the larvae body was fully covered by melanophores; when analysis was performed. This indicates that the allostatic load exerted on fish of different groups was similar and inside the fish-coping abilities range, in terms of the cortisol response axis. The selection of the experimental scale is of importance, especially when the results are to be transferred and applied on an industrial scale.

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 β2 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.