Characterization and Expression Dynamics of Key Genes Involved in the Gilthead Sea Bream (Sparus aurata) Cortisol Stress Response during Early Ontogeny.

The present study identified and characterized six key genes involved in the hypothalamic-pituitary-interrenal (HPI) axis of gilthead sea bream (Sparus aurata), a commercially important European aquaculture species. The key genes involved in the HPI axis for which gene structure and synteny analysis was carried out, comprised of two functional forms of glucocorticoid receptors (GR), as well as three forms of pro-opiomelanocortin (POMC) genes and one form of mineralocorticoid receptor (MR) gene. To explore their functional roles during development but also in the stress response, the expression profiles of gr1, gr2, mr, pomc_aI, pomc_aII, and pomc_ß were examined during early ontogeny and after an acute stress challenge. The acute stress challenge was applied at the stage of full formation of all fins, where whole body cortisol was also measured. Both the cortisol and the molecular data implied that sea bream larvae at the stage of the full formation of all fins at 45 dph are capable of a response to stress of a similar profile as observed in adult fish.

The transcriptomic signature of different sexes in two protogynous hermaphrodites: Insights into the molecular network underlying sex phenotype in fish.

Sex differentiation is a puzzling problem in fish due to the variety of reproductive systems and the flexibility of their sex determination mechanisms. The Sparidae, a teleost family, reflects this remarkable diversity of sexual mechanisms found in fish. Our aim was to capture the transcriptomic signature of different sexes in two protogynous hermaphrodite sparids, the common pandora Pagellus erythrinus and the red porgy Pagrus pagrus in order to shed light on the molecular network contributing to either the female or the male phenotype in these organisms. Through RNA sequencing, we investigated sex-specific differences in gene expression in both species' brains and gonads. The analysis revealed common male and female specific genes/pathways between these protogynous fish. Whereas limited sex differences found in the brain indicate a sexually plastic tissue, in contrast, the great amount of sex-biased genes observed in gonads reflects the functional divergence of the transformed tissue to either its male or female character. Α common "crew" of well-known molecular players is acting to preserve either sex identity of the gonad in these fish. Lastly, this study lays the ground for a deeper understanding of the complex process of sex differentiation in two species with an evolutionary significant reproductive system.

α-MSH and melanocortin receptors at early ontogeny in European sea bass (Dicentrarchus labrax, L.).

Temporal patterns of whole-body α-MSH concentrations and of transcripts of melanocortin receptors during early development as well as the endocrine response (α-MSH, cortisol, MCR mRNAs) to stress at the end of the larval period were characterized in Dicentrarchus labrax. Immunohistochemistry showed α-MSH positive cells in the pituitary pars intermedia in all stages examined. As development proceeds, α-MSH content gradually increases; mRNA levels of mc2r and mc4r remain low until first feeding where peak values are observed. Mc1r expression was constant during development, pomc mRNA levels remain low until the stage of flexion after which a significant increase is observed. At the stage of the formation of all fins, whole-body cortisol and α-MSH concentrations responded with peak values at 2 h post stress. Additionally, the stress challenge resulted in elevated transcript levels of pomc, mc2r and mc4r but not in mc1r, with a pattern characterized by peak values at 1 h post stress and a strong correlation with whole body α-MSH concentrations was found. Our data provide for the first time a view on the importance of the α-MSH stress response in early development of European sea bass, an additional and relatively poorly understood signal involved in the stress response in teleosts.

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

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.

Transcriptomic changes in relation to early-life events in the gilthead sea bream (Sparus aurata).

BACKGROUND: Teleosts are exposed to a broad range of external stimuli, which may be either of acute or chronic nature. The larval phase of certain fish species offer a unique opportunity to study the interactions between genes and environmental factors during early life. The present study investigates the effects of early-life events, applied at different time points of early ontogeny (first feeding, flexion and development of all fins; Phase 1) as well as on the subsequent juvenile stage after the application of an additional acute stressor (Phase 2) in the gilthead sea bream (Sparus aurata), a commercially important European aquaculture species. Animal performance, the cortisol response and gene expression patterns during early development as well as on the subsequent phases (juveniles) after the application of additional acute stressors were investigated. RESULTS: Significant differences on fish performance were found only for juveniles exposed to early-life events at the phase of the formation of all fins. On the transcriptome level distinct expression patterns were obtained for larvae as well as for juveniles with the most divergent expression pattern found to be again at the phase of the development of all fins, which showed to have also an impact later on in the acute stress response of juveniles. CONCLUSIONS: The present study showed that applying an early-life protocol, characterized by the unpredictable, variable and moderate intensity of the applied stimuli provides a relative realistic model to evaluate the impact of daily aquaculture practices on fish performance. In addition, the power of investigating global gene expression patterns is shown, providing significant insights regarding the response of early-life events during development and as juveniles after the application of extra acute stressors.

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.

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 11ß-hydroxylase and 11ß-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.

Diel changes in plasma cortisol and effects of size and stress duration on the cortisol response in European sea bass (Dicentrarchus labrax).

European sea bass (Dicentrarchus labrax), one of the most economically important fish in Mediterranean mariculture, shows high basal cortisol concentrations compared with other teleosts. The present study aims (a) to identify cortisol diel variation in fish held under a 12L:12D cycle and minimum handling stress, and (b) to establish the effect of fish size and stressor duration on the cortisol response. The results indicate high intrapopulation variability in plasma cortisol and a significant diel fluctuation with a peak value at dusk (18 h). Stressors of different intensity and/or duration affected the cortisol stress response in a differential manner according to fish size (and/or age). Maximum cortisol values in small-size fish were found at 1 and 2 h post-stress, depending on the duration of the stressor, while at 0.5 h post-stress in large fish regardless stress duration.

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.

Ontogeny of the thyroid hormones and cortisol in the gilthead sea bream, Sparus aurata.

The aim of the study was to elucidate the pattern of changes in the whole body concentrations of thyroid hormones (TH) and cortisol in eggs, pre-larvae, and larvae in the gilthead sea bream, Sparus aurata. The ontogeny of these hormones was related to specific morphological characteristics that characterize early development. TH and cortisol were detected in substantial amounts in the eggs of sea bream. T3 concentration in eggs was found to be higher than that of T4 (T3, 4.02+/-0.38 ng g-1; T4, 1.63+/-0.28 ng g-1), as it is often observed in marine teleosts. T4 decreased until hatching (T4, 1.01+/-0.58 ng g-1), while T3 reached a minimum at the time of first feeding (T3, 0.19+/-0.02 ng g-1). As development proceeded, both hormones displayed a similar pattern of changes with increasing concentrations until the completion of metamorphosis (T3, 15.84+/-2.92 ng g-1; T4, 9.78+/-2.39 ng g-1), indicating that the hypothalamo-adenohypophysial-thyroid axis begins to function soon after first feeding. The pattern of changes of cortisol (F) concentration was almost parallel to that of T4, starting from 0.83 ng g-1 in eggs. Minimum F concentration was observed at hatching (0.39+/-0.03 ng g-1) and maximum at melanophores and scale formation (14.82+/-2.71 and 14.82+/-3.12 ng g-1, respectively). Results provide data for the first time on the ontogeny of thyroid hormones and cortisol during the early development in sea bream and are in agreement with results in other fish species for an important action of TH during early development.