Stress response of Rhamdia quelen to the interaction stocking density - Feeding regimen.

This study aimed to verify the effect of different feeding and stocking conditions during 14 days on the gene expression of several hormones and enzymes related to the stress cascade and metabolic parameters in silver catfish Rhamdia quelen under the following experimental conditions: 1) fed at low stocking density (2.5 kg m-3, LSD-F); 2) fed at high stocking density (32 kg m-3, HSD-F); 3) food-deprived at LSD (LSD-FD); and 4) food-deprived at HSD (HSD-FD). Fish from LSD-F and HSD-F groups were fed daily (1 % of their body mass), while fish from food-deprived groups (LSD-FD and HSD-FD) were not fed during the experimental time. Plasma metabolic parameters (glucose, lactate, triglycerides, and proteins) and hepatosomatic index (HSI) were evaluated. In addition, mRNA expression of genes related to the stress axis (crh, pomca, pomcb, nr3c2, star, hsd11b2 and hsd20b), heat shock protein family (hsp90 and hspa12a), sodium-dependent noradrenaline transporter (slc6a2), and growth axis (gh and igf1) were also assessed. Specific growth rate and HSI decreased in food-deprived fish regardless of stocking density. The HSD-FD group showed weight loss compared to the HSD-F, LSD-F, and LSD-FD groups. Plasma glucose and triglycerides were reduced in food-deprived groups, while lactate and protein levels did not change. The expression of key players of the stress response (crh, pomca, pomcb, hsd11b2, nr3c2, and hsp90b) and growth (gh and igf1) pathways were differently regulated depending on the experimental condition, whereas no statistical difference between treatments was found for hsd20b, scl6a2, hspa12a, and star mRNAs expression. This study suggests that LSD acts as a stressor affecting negatively the physiological status of fed fish, as demonstrated by the reduction in growth rates, altered metabolic orchestration, and a higher crh mRNA expression. In addition, food deprivation also increased mRNA expression of other assessed genes (nr3c2, hsp90b, pomca, and pomcb) in fish from the HSD group, indicating higher responsiveness to stress in this stocking density when combined with food deprivation.

Osmoregulatory role of vasotocinergic and isotocinergic systems in the gilthead sea bream (Sparus aurata L).

Gilthead sea bream, Sparus aurata L., is an important fish species for the Mediterranean aquaculture and is considered a good model for studying the osmoregulatory process, due to its capacity to cope with great changes in environmental salinity (5-60‰). Our group studied the osmoregulatory role of different endocrine systems in this species, focusing on the vasotocinergic and isotocinergic systems over several years. For this purpose, the cDNAs coding for pro-vasotocin (pro-vt), pro-isotocin (pro-it), two arginine vasotocin (AVT) receptors (avtr v1a2- and v2-types) and one IT receptor (itr) were cloned. Acclimation to different environmental salinities induced a direct lineal relationship between plasma AVT levels and salinity, with no changes in plasma IT values. In addition, higher values in vasotocinergic, isotocinergic and stress pathways (pro-vt and pro-it gene expression, AVT and IT storage and plasma cortisol levels) in both hypo- and/or hyper-osmotic transfers, suggest an interaction between cortisol and AVT/IT pathways. Moreover, gene expression of specific receptors, as well as the use of different in vitro techniques, demonstrated an important osmoregulatory orchestration in different organs. In addition, individuals intraperitoneally injected with AVT and transferred to different environmental salinities enhanced plasma cortisol levels and/or gill Na+, K+-ATPase activity. These effects could be related to the energy repartitioning process occurring during osmotic adaptation of S. aurata to extreme environmental salinities, which could be mediated not only by plasma cortisol but also by AVT. Finally, our results indicated a very important role of the vasotocinergic and/or isotocinergic systems in both osmoregulatory and non-osmoregulatory organs.

Unraveling vasotocinergic, isotocinergic and stress pathways after food deprivation and high stocking density in the gilthead sea bream.

The influence of chronic stress, induced by food deprivation (FD) and/or high stocking density (HSD), was assessed on stress, vasotocinergic and isotocinergic pathways of the gilthead sea bream (Sparus aurata). Fish were randomly assigned to one of the following treatments: (1) fed at low stocking density (LSD-F; 5kg·m-3); (2) fed at high stocking density (HSD-F, 40kg·m-3); (3) food-deprived at LSD (LSD-FD); and (4) food-deprived at HSD (HSD-FD). After 21days, samples from plasma, liver, hypothalamus, pituitary and head-kidney were collected. Both stressors (FD and HSD) induced a chronic stress situation, as indicated by the elevated cortisol levels, the enhancement in corticotrophin releasing hormone (crh) expression and the down-regulation in corticotrophin releasing hormone binding protein (crhbp) expression. Changes in plasma and liver metabolites confirmed a metabolic adjustment to cope with energy demand imposed by stressors. Changes in avt and it gene expression, as well as in their specific receptors (avtrv1a, avtrv2 and itr) at central (hypothalamus and pituitary) and peripheral (liver and head-kidney) levels, showed that vasotocinergic and isotocinergic pathways are involved in physiological changes induced by FD or HSD, suggesting that different stressors are handled through different stress pathways in S. aurata.

Gene expression profiling of whole blood cells supports a more efficient mitochondrial respiration in hypoxia-challenged gilthead sea bream (Sparus aurata).

BACKGROUND: Acclimation to abiotic challenges, including decreases in O2 availability, requires physiological and anatomical phenotyping to accommodate the organism to the environmental conditions. The retention of a nucleus and functional mitochondria in mature fish red blood cells makes blood a promising tissue to analyse the transcriptome and metabolic responses of hypoxia-challenged fish in an integrative and non-invasive manner. METHODS: Juvenile gilthead sea bream (Sparus aurata) were reared at 20-21 °C under normoxic conditions (> 85% O2 saturation) followed by exposure to a gradual decrease in water O2 concentration to 3.0 ppm (41-42% O2 saturation) for 24 h or 1.3 ppm (18-19% O2 saturation) for up to 4 h. Blood samples were collected at three different sampling points for haematological, biochemical and transcriptomic analysis. RESULTS: Blood physiological hallmarks remained almost unaltered at 3.0 ppm, but the haematocrit and circulating levels of haemoglobin, glucose and lactate were consistently increased when fish were maintained below the limiting oxygen saturation at 1.3 ppm. These findings were concurrent with an increase in total plasma antioxidant activity and plasma cortisol levels, whereas the opposite trend was observed for growth-promoting factors, such as insulin-like growth factor I. Additionally, gene expression profiling of whole blood cells revealed changes in upstream master regulators of mitochondria (pgcß and nrf1), antioxidant enzymes (gpx1, gst3, and sod2), outer and inner membrane translocases (tom70, tom22, tim44, tim10, and tim9), components of the mitochondrial dynamics system (mfn2, miffb, miro1a, and miro2), apoptotic factors (aifm1), uncoupling proteins (ucp2) and oxidative enzymes of fatty acid ß-oxidation (acca2, ech, and hadh), the tricarboxylic acid cycle (cs) and the oxidative phosphorylation pathway. The overall response is an extensive reduction in gene expression of almost all respiratory chain enzyme subunits of the five complexes, although mitochondrial-encoded catalytic subunits and nuclear-encoded regulatory subunits of Complex IV were primarily increased in hypoxic fish. CONCLUSIONS: Our results demonstrate the re-adjustment of mitochondrial machinery at transcriptional level to cope with a decreased basal metabolic rate, consistent with a low risk of oxidative stress, diminished aerobic ATP production and higher O2-carrying capacity. Taken together, these results suggest that whole blood cells can be used as a highly informative target tissue of metabolic condition.

Molecular performance of Prl and Gh/Igf1 axis in the Mediterranean meager, Argyrosomus regius, acclimated to different rearing salinities.

Aquaculture industry in the Mediterranean region exhibits a growing interest for the Mediterranean meager Argyrosomus regius. Some preliminary works showed a good growth performance of the species in nearly isosmotic salinities. However, the patterns of alteration of prolactin (Prl) as well as growth hormone (Gh)/insulin growth factor-1 (Igf1) axis at the molecular level are not yet described in this species. Therefore, we cloned and sequenced partial cDNAs for pituitary prolactin (prl) and growth hormone (gh), hepatic insulin-like growth factor (igf1), and ß-actin (actb). Expression patterns of these transcripts were tested in juveniles of A. regius acclimated to four different environmental salinities: (1) 5 ‰ (hyposmotic); (2) 12 ‰ (isosmotic); (3) 38 ‰ (hyperosmotic; seawater control); and (4) 55 ‰ (extremely hyperosmotic). All investigated transcripts shared high sequence identities with their counterparts in other perciformes. prl mRNA levels showed inverse pattern with increasing salinities. gh mRNA enhanced significantly in both 12 and 55 ‰ salinity groups in comparison with the control group, while igf1 showed its maximum expression levels under the nearly isosmotic environment. The results indicated clear sensitivity of prl, gh and igf1 to changes in environmental salinity, which can possibly control the euryhalinity capacity of this species.

Vasotocin and isotocin regulate aquaporin 1 function in the sea bream.

Aquaporins (AQPs) are specific transmembrane water channels with an important function in water homeostasis. In terrestrial vertebrates, AQP2 function is regulated by vasopressin (AVP) to accomplish key functions in osmoregulation. The endocrine control of aquaporin function in teleosts remains little studied. Therefore, in this study we investigated the regulatory role of vasotocin (AVTR) and isotocin (ITR) receptors in Aqp1 paralog gene function in the teleost gilthead sea bream (Sparus aurata). The complete coding regions of Aqp1a, Aqp1b, AVTR V1a2-type, AVTR V2-type and ITR from sea bream were isolated. A Xenopus oocyte-swelling assay was used to functionally characterize AQP1 function and regulation by AVT and IT through their cognate receptors. Microinjection of oocytes with Aqp1b mRNA revealed regulation of water transport via PKA (IBMX+forskolin sensitive), whereas Aqp1a mRNA injection had the same effect via PKC signaling (PDBU sensitive). In the absence of expressed receptors, AVT and IT (10(-8) mol l(-1)) were unable to modify AQP1 function. AVT regulated AQP1a and AQP1b function only when the AVTR V2-type was co-expressed. IT regulated AQP1a function, but not AQP1b, only when ITR was present. Considering that Aqp1a and Aqp1b gene expression in the sea bream intestine is highly salinity dependent in vivo, our results in ovo demonstrate a regulatory role for AVT and IT in AQP1 function in the sea bream in the processing of intestinal fluid to achieve osmoregulation.

AVT and IT regulate ion transport across the opercular epithelium of killifish (Fundulus heteroclitus) and gilthead sea bream (Sparus aurata).

The regulatory role of arginine vasotocin (AVT) and isotocin (IT) in Cl(-) secretion was investigated with the short circuit current (Isc) technique in opercular epithelia of killifish (Fundulus heteroclitus) and gilthead sea bream (Sparus aurata). Sea bream operculum showed ~4-fold lower number of Na/K-ATPase immunoreactive cells and ~12-fold lower secretory current than the killifish. In sea bream opercular membranes, the basolateral addition of AVT (10(-6) M) significantly stimulated Cl(-) secretion, while IT (10(-6) M) was without effect. In killifish, IT produced an immediate dose-dependent stimulation of Cl(-) secretion with significant effect at doses ≥10(-7) M and stimulation maxima (∆Isc ~25 µA⋅cm(-2)) at 10(-6) M. The basolateral addition of bumetanide (200 µM) abolished >75% of the effect of IT on Cl(-) secretion. In turn, AVT had a dual effect on killifish opercular Isc: an immediate response (~3min) with Isc reduction in an inverted bell-shaped dose-response manner with higher current decrease (-22 µA⋅cm(-2)) at 10(-8) M AVT, and a sustained dose-dependent stimulation of Cl(-) secretion (stable up to 1h), with a threshold significant effect at 10(-8) M and maximal stimulation (~20 µA⋅cm(-2)) at 10(-6)M. Both effects of AVT appear receptor type specific. The V1-receptor antagonist SR 49059 abolished Isc reduction in response to AVT, while the specific V2-receptor antagonist (Tolvaptan, 1 µM) abolished the stimulatory action of AVT on Cl(-) secretion. According to these results, we propose a modulatory role for AVT and IT in Cl(-) (NaCl) secretion across the opercular epithelium of marine teleost.

Stress response in silver catfish (Rhamdia quelen) exposed to the essential oil of Hesperozygis ringens.

This study investigated the effects of prolonged exposure of silver catfish (Rhamdia quelen) to the essential oil (EO) of Hesperozygis ringens. Ventilatory rate (VR), stress and metabolic indicators, energy enzyme activities, and mRNA expression of adenohypophyseal hormones were examined in specimens that were exposed for 6 h to 0 (control), 30 or 50 µL L(-1) EO of H. ringens in water. Reduction in VR was observed in response to each treatment, but no differences were found between treatments. Plasma glucose, protein, and osmolality increased in fish exposed to 50 µL L(-1). Moreover, lactate levels increased after exposure to both EO concentrations. Plasma cortisol levels were not changed by EO exposure. Fish exposed to 30 µL L(-1) EO exhibited higher glycerol-3-phosphate dehydrogenase (G3PDH) activity, while exposure to 50 µL L(-1) EO elicited an increase in glucose-6-phosphate dehydrogenase (G6PDH), fructose-biphosphatase (FBP), and 3-hydroxyacyl-CoA-dehydrogenase (HOAD) activities compared with the control group. Expression of growth hormone (GH) only decreased in fish exposed to 50 µL L(-1) EO, while somatolactin (SL) expression decreased in fish exposed to both concentrations of EO. Exposure to EO did not change prolactin expression. The results indicate that GH and SL are associated with energy reorganization in silver catfish. Fish were only slightly affected by 30 µL L(-1) EO of H. ringens, suggesting that it could be used in practices where a reduction in the movement of fish for prolonged periods is beneficial, i.e., such as during fish transportation.

Tissue explants as tools for studying the epigenetic modulation of the GH-IGF-I axis in farmed fish.

Somatic growth in vertebrates is mainly controlled by the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. The role of epigenetic mechanisms in regulating this axis in fish is far from being understood. This work aimed to optimize and evaluate the use of short-term culture of pituitary and liver explants from a farmed fish, the gilthead seabream Sparus aurata, for studying epigenetic mechanisms involved in GH/IGF-I axis regulation. Our results on viability, structure, proliferation, and functionality of explants support their use in short-term assays. Pituitary explants showed no variation in gh expression after exposure to the DNA methylation inhibitor decitabine (5-Aza-2'-deoxycytidine; DAC), despite responding to DAC by changing dnmt3bb and tet1 expression, and TET activity, producing an increase in overall DNA hydroxymethylation. Conversely, in liver explants, DAC had no effects on dnmt s and tet s expression or activity, but modified the expression of genes from the GH-IGF-I axis. In particular, the expression of igfbp2a was increased and that of igfbp4, ghri and ghrii was decreased by DAC as well as by genistein, which is suggestive of impaired growth. While incubation of liver explants with S-adenosylmethionine (SAM) produced no clear effects, it is proposed that nutrients must ensure the methylation milieu within the liver in the fish to sustain proper growth, which need further in vivo verification. Pituitary and liver explants from S. aurata can be further used as described herein for the screening of inhibitors or activators of epigenetic regulators, as well as for assessing epigenetic mechanisms behind GH-IGF-I variation in farmed fish.

AVT is involved in the regulation of ion transport in the intestine of the sea bream (Sparus aurata).

The intestine of marine fish plays a crucial role in ion homeostasis by selective processing of ingested fluid. Although arginine vasotocin (AVT) is suggested to play a role in ion regulation in fish, its action in the intestine has not been demonstrated. Thus, the present study investigated in vitro the putative role of AVT in intestinal ion transport in the sea bream (Sparus aurata). A cDNA encoding part of an AVT receptor was isolated and phylogenetic analysis revealed it clustered with the V1a2-type receptor clade. V1a2 transcripts were expressed throughout the gastrointestinal tract, from esophagus to rectum, and were most abundant in the rectum regardless of long-term exposure to external salinities of 12, 35 or 55p.p.t. Basolateral addition of AVT (10(-6)M) to the anterior intestine and rectum of sea bream adapted to 12, 35 or 55p.p.t. mounted in Ussing chambers produced rapid salinity and region dependent responses in short circuit current (Isc), always in the absorptive direction. In addition, AVT stimulation of absorptive Isc conformed to a dose-response curve, with significant effects achieved at 10(-8)M, which corresponds to physiological values of plasma AVT for this species. The effect of AVT on intestinal Isc was insensitive to the CFTR selective inhibitor NPPB (200µM) applied apically, but was completely abolished in the presence of apical bumetanide (200µM). We propose a role for AVT in the regulation of ion absorption in the intestine of the sea bream mediated by an absorptive bumetanide-sensitive mechanism, likely NKCC2.

Assessing differences in the bioaccessibility of phenolics present in two wine by-products using an in-vitro model of fish digestion.

Increasing attention is currently being paid to the protective role of polyphenols in health and oxidative status in fish. For this reason, the potential use of different natural sources of such compounds, like wine by products, is under study. One key step required to gain a better understanding on the biological roles of polyphenols for a given species is to assess the different factors affecting their digestive bioaccessibility, and a great number of such studies is based in the use of in vitro digestion models. In the present study the potential digestive bioavailability of the phenolic compounds present in wine bagasse and lees was evaluated for two fish species showing great differences in their digestive phisyiology: the omnivorous gilthead sea bream (Sparus aurata) and the herbivorous flathead grey mullet (Mugil cephalus). The study was developed using in vitro models adapted to simulate their digestion and a factorial experimental design that simultaneously evaluated the effects of the ingredient used as source of polyphenols, presence or absence of feed matrix, fish species and digestion time. The release of the phenolic compounds was evaluated using ultra-high performance liquid chromatography (UHPLC) coupled to high resolution mass spectrometry (HRMS) detection. Both the presence of feed matrix and the type of wine by-product showed a significant effect on the digestive release of both total and specific types of polyphenols while fish species showed to be significant only for some specific compounds, like eriodyctiol or syringic acid. The time of digestion was not identified as a statistically significant factor in the release of phenolic compounds due to the great variability in the patterns observed that were classified as early, sustained and late. The observed great variations in the patterns of release of different types of phenolic compounds with time suggest an important effect of gut transit rates on the net bioavailability of a given phenolic compound in the live fish. The present study is, to our knowledge, the first one on which an in vitro approach was applied to assess to what extent the possible complexation of wine polyphenols present in wine by-products with either digestive enzymes or components of the feed matrix could limit their bioaccessibility if included in diets of two different fish species.

Evaluation of the Combined Administration of Chlorella fusca and Vibrio proteolyticus in Diets for Chelon labrosus: Effects on Growth, Metabolism, and Digestive Functionality.

This study aimed to evaluate the combined effect of dietary Chlorella fusca and ethanol-inactivated Vibrio proteolyticus DCF12.2 (C + V diet) in Chelon labrosus juveniles, highlighting their nutritional, physiological, and morphological effects. The results showed that the combined dietary inclusion of C. fusca and V. proteolyticus significantly enhanced growth performance and feed utilization compared to the control group. The C + V diet increased the fish lipid quality index (FLQ), n-3 polyunsaturated fatty acids, and n-3/n-6 ratio, which might be beneficial in terms of human nutrition. The C + V diet considerably increased carbohydrate metabolic activity by statistically boosting plasma glucose. The dietary inclusion of C. fusca in conjunction with V. proteolyticus increased metabolic enzyme activity as well as intestinal absorption capacity compared to that found in the control group. In conclusion, the experimental diet was suitable for feeding C. labrosus, increasing their growth and the nutritional characteristics of the muscle and intestine, without causing tissue damage.

High Stocking Density and Food Deprivation Increase Brain Monoaminergic Activity in Gilthead Sea Bream (Sparus aurata).

In teleosts, brain monoamines (dopamine and serotonin) participate in the early response to different acute stressors. However, little is known regarding their role during chronic stress. In a 2 × 2 factorial design, the influence of a high stocking density (HSD) and/or food deprivation (FD) on the brain monoaminergic activity in gilthead sea bream (Sparus aurata) was evaluated. Following a 21-day experimental design, samples from the plasma and brain regions (telencephalon, hypothalamus, and optic tectum) were collected. The dopamine (DA), serotonin (5HT), and their main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5 hydroxyindoleacetic acid (5HIAA), contents were HPLC-assessed in brain tissues, and the ratios DOPAC/DA and 5HIAA/5HT were calculated as indicators of enhanced monoaminergic activity. The plasma levels of cortisol and catecholamine were also evaluated. The cortisol levels increased in fish exposed to HSD and normally fed but, also, in all FD groups, whereas the NA levels decreased in LSD-FD animals. Within the brain, the dopaminergic and serotonergic activities in telencephalon and hypothalamus increased in fish subjected to HSD and in the telencephalon of LSD-FD fish. While DA (hypothalamus) and 5HT (telencephalon) increased in the animals submitted to a HSD, food-deprived fish did not show such an increase. Taken together, our results supported the hypothesis of brain monoaminergic activity participating in maintaining and orchestrating the endocrine response to chronic stress in fish.

Ontogeny of Expression and Activity of Digestive Enzymes and Establishment of gh/igf1 Axis in the Omnivorous Fish Chelon labrosus.

Thick-lipped grey mullet (Chelon labrosus) is a candidate for sustainable aquaculture due to its omnivorous/detritivorous feeding habit. This work aimed to evaluate its digestive and growth potentials from larval to early juvenile stages. To attain these objectives the activity of key digestive enzymes was measured from three until 90 days post hatch (dph). Expression of genes involved in digestion of proteins (try2, ctr, pga2, and atp4a), carbohydrates (amy2a), and lipids (cel and pla2g1b), together with two somatotropic factors (gh and igf1) were also quantified. No chymotrypsin or pepsin activities were detected. While specific activity of trypsin and lipase were high during the first 30 dph and declined afterward, amylase activity was low until 57 dph and increased significantly beyond that point. Expression of try2, ctr, amy2a, and cel increased continuously along development, and showed a peak at the end of metamorphosis. Expression of pla2g1b, pga2 and atp4a increased until the middle of metamorphosis and decreased afterwars. Most of these trends contrast the usual patterns in carnivorous species and highlight the transition from larvae, with high protein requirements, to post-larvae/juvenile stages, with omnivorous/detritivorous feeding preferences. Somatotropic genes, gh and igf1, showed approximately inverse expression patterns, suggesting the establishment of the Gh/Igf1 axis from 50 dph.

Low dietary inclusion of nutraceuticals from microalgae improves feed efficiency and modifies intermediary metabolisms in gilthead sea bream (Sparus aurata).

The aim of this work was to evaluate two functional feeds for the gilthead seabream, Sparus aurata, containing low inclusion of two microalgae-based products (LB-GREENboost, LBGb; and LB-GUThealth, LBGh). Fish (12-13 g) were fed for 13 weeks a control diet or one of the four diets supplemented with both products at 0.5% or 1%. LBGb and LBGh did not affect specific growth rate or survival, but increased feed efficiency by decreasing feed intake and enlarging the intestines. LBGb increased hepatosomatic index and reduced cortisol levels in plasma, while both products lowered plasma lactate. Extensive metabolite and metabolic enzyme profiling revealed that microalgae supplementations, especially 1% LBGh: (i) decrease plasma lactate and increase hepatic glycogen, (ii) reduce hepatic gluconeogenesis, (iii) enhance hepatic lipogenic activity and lipid secretion, (iv) led fish to double triglyceride content in muscle and to stimulate its lipid oxidative capacity, and (v) increase the content of monounsaturated fatty acids and the omega-3 alpha-linolenic acid in muscle. This study demonstrates that both microalgae-based products are suited to improve feed efficiency and orchestrate significant changes in the intermediary metabolism in gilthead seabream juveniles.

Essential Oils as Stress-Reducing Agents for Fish Aquaculture: A Review.

In fish, stressful events initiate a hormone cascade along the hypothalamus-pituitary-interrenal and hypothalamus-sympathetic-chromaffin (HSC) axis to evoke several physiological reactions in order to orchestrate and maintain homeostasis. Several biotic and abiotic factors, as well as aquaculture procedures (handling, transport, or stocking density), activated stress system inducing negative effects on different physiological processes in fish (growth, reproduction, and immunity). In order to reduce these consequences, the use of essential oils (EOs) derived from plants has been the focus of aquaculture studies due to their diverse properties (e.g., anesthetic, antioxidant, and antimicrobial), which have been shown to reduce biochemical and endocrine alterations and, consequently, to improve the welfare status. Recently, several studies have shown that biogenic compounds isolated from different EOs present excellent biological activities, as well as the nanoencapsulated form of these EOs may potentiate their effects. Overall, EOs presented less side effects than synthetic compounds, but their stress-reducing efficacy is related to their chemical composition, concentration or chemotype used. In addition, their species-specific actions must be clearly established since they can act as stressors by themselves if their concentrations and chemotypes used are not suitable. For this reason, it is necessary to assess the effect of these natural compound mixtures in different fish species, from marine to freshwater, in order to find the ideal concentration range and the way for their administration to obtain the desired biological activity, without any undesired side effects. In this review, the main findings regarding the use of different EOs as stress reducers will be presented to highlight the most important issues related to their use to improve fish welfare in aquaculture.

Tissue-Specific Orchestration of Gilthead Sea Bream Resilience to Hypoxia and High Stocking Density.

Two different O2 levels (normoxia: 75-85% O2 saturation; moderate hypoxia: 42-43% O2 saturation) and stocking densities (LD: 9.5, and HD: 19 kg/m3) were assessed on gilthead sea bream (Sparus aurata) in a 3-week feeding trial. Reduced O2 availability had a negative impact on feed intake and growth rates, which was exacerbated by HD despite of the improvement in feed efficiency. Blood physiological hallmarks disclosed the enhancement in O2-carrying capacity in fish maintained under moderate hypoxia. This feature was related to a hypo-metabolic state to cope with a chronic and widespread environmental O2 reduction, which was accompanied by a differential regulation of circulating cortisol and growth hormone levels. Customized PCR-arrays were used for the simultaneous gene expression profiling of 34-44 selected stress and metabolic markers in liver, white skeletal muscle, heart, and blood cells. The number of differentially expressed genes ranged between 22 and 19 in liver, heart, and white skeletal muscle to 5 in total blood cells. Partial Least-Squares Discriminant Analysis (PLS-DA) explained [R2Y(cum)] and predicted [Q2Y(cum)] up to 95 and 65% of total variance, respectively. The first component (R2Y = 0.2889) gathered fish on the basis of O2 availability, and liver and cardiac genes on the category of energy sensing and oxidative metabolism (cs, hif-1α, pgc1α, pgc1ß, sirts 1-2-4-5-6-7), antioxidant defense and tissue repair (prdx5, sod2, mortalin, gpx4, gr, grp-170, and prdx3) and oxidative phosphorylation (nd2, nd5, and coxi) highly contributed to this separation. The second component (R2Y = 0.2927) differentiated normoxic fish at different stocking densities, and the white muscle clearly promoted this separation by a high over-representation of genes related to GH/IGF system (ghr-i, igfbp6b, igfbp5b, insr, igfbp3, and igf-i). The third component (R2Y = 0.2542) discriminated the effect of stocking density in fish exposed to moderate hypoxia by means of hepatic fatty acid desaturases (fads2, scd1a, and scd1b) and muscle markers of fatty acid oxidation (cpt1a). All these findings disclose the different contribution of analyzed tissues (liver ≥ heart > muscle > blood) and specific genes to the hypoxic- and crowding stress-mediated responses. This study will contribute to better explain and understand the different stress resilience of farmed fish across individuals and species.

Arginine Vasotocin and Cortisol Co-regulate Vasotocinergic, Isotocinergic, Stress, and Thyroid Pathways in the Gilthead Sea Bream (Sparus aurata).

In teleosts, a complex interaction between several endocrine axes modulates physiological functions related to metabolism, stress, and osmoregulation. Although many studies in fish underline the interconnection between the hypothalamic-pituitary-interrenal (HPI) and hypothalamic-pituitary-thyroid (HPT) endocrine axes, their relationship with the vasotocinergic and isotocinergic systems remains unknown. The aim of the present study is therefore to shed light on the potential cross-regulations between HPT, HPI, and the vasotocinergic and isotocinergic axes in gilthead sea bream (Sparus aurata) at hypothalamic, hypophyseal, and plasma levels. Sea breams were administered with intraperitoneal slow-release implants containing different doses of vasotocin (the active peptide in vasotocinergic system) or cortisol (the last component of HPI axis). Plasma osmolality was higher in active neuropeptides vasotocin (Avt)-treated fish, indicating an osmoregulatory function of this hormone. Low concentrations of Avt increased hypothalamic arginine vasotocin precursor (avt) mRNA levels and increased Avt storage in the pituitary. Avt treatment down-regulated hypothalamic arginine vasotocin receptor v1a-type (avtrv1a), suggesting a negative paracrine co-regulation of the HPI axis due to the close location of avtrv1a and adrenocorticotropin hormone (Acth) cells in the anterior pituitary. Furthermore, the up-regulation observed in arginine vasotocin receptor v2-type (avtrv2) suggests their involvement in metabolic and cortisol-related pathways in the hypothalamus. The decrease in isotocin (It) pituitary storage and the up-regulation of it receptor, observed in the Avt-treated group, reinforce the idea of an interconnection between the vasotocinergic and isotocinergic systems. Cortisol and Avt administration each inhibited the HPI axis, down-regulating crh gene expression in the absence of variations in corticotropin releasing hormone binding protein (crhbp). Finally, both hormonal treatments activated the HPT axis via up-regulation of trh and down-regulation of thrb. Our results provide evidence for strong interactions among the Avt/It, HPI, and HPT axes of marine teleosts, particularly at the hypothalamic level.

Molecular basis of the digestive functionality in developing Persian sturgeon (Acipenser persicus) larvae: additional clues for its phylogenetic status.

Persian sturgeon (Acipenser persicus) is a critically endangered species, mainly due to overexploitation for its caviar. The permanence of populations of this species in the Caspian Sea is fully dependent on restocking programs. Accordingly, it is considered as an interesting target for aquaculture for both restocking and commercial purposes. In addition, as a Chondrostei, it exhibits one of the slowest rates of molecular evolution among vertebrates and is propounded as an excellent candidate for phylogenetic analysis and evolutionary biology. In this study, the early ontogeny of some key digestive enzymes precursors was determined at molecular level, aiming to obtain basic knowledge on the acquisition of digestive capacity of this species, and at the same time, to advance in its phylogenetic status from the point of view of digestion. For this purpose, A. persicus cDNAs for ß-actin (actb; used as an internal reference gene), bile-salt activated lipase (cel), trypsinogen 1 (try1), pepsinogen (pga), and gastric proton pump (atp4a) were amplified and cloned, and their subsequent expressions were measured by quantitative real-time PCR during the first 34 days post hatch (dph). Two isoforms for pga and at least six for try1 were obtained in this study, probably due to the additional genome duplication which sturgeons suffered along evolution. Phylogenetic analysis of the deduced amino acids sequences from the studied genes demonstrated that this species has a close evolutionary distance to Holostei, coelacanths, and tetrapods, including amphibians, reptiles, birds and mammals. According to our results, expression of all the genes increased gradually over time and reached maximum levels around 18 dph. This pattern, which was comparable to length and weight data, could indicate that around 3 weeks after hatching, the digestive capacity of the Persian sturgeon changes from larval to juvenile mode.

Aroclor 1254 inhibits vasotocinergic pathways related to osmoregulatory and stress functions in the gilthead sea bream (Sparus aurata, Linnaeus 1758).

The present study assesses the response of vasotocinergic system in the gilthead sea bream (Sparus aurata) after administering two doses of the polychlorinated biphenyl Aroclor 1254 (15 or 50 µg g-1 fresh body mass). Seven days post-administration, eight fish of each experimental group were sampled, and the remaining animals were challenged with a hyperosmotic stress by being transferred from seawater (36 ppt) to high salinity water (55 ppt) and being sampled 3 days post-transfer. Aroclor 1254 affected gene expression of avt, together with Avt concentrations in pituitary and plasma, inhibiting the stimulation observed in vasotocinergic system after hyperosmotic challenge. This was noted by the accumulation of Avt at hypophyseal level as well as by its undetectable values in plasma. Hyperosmotic transfer significantly changed branchial avtrv1a, avtrv2, atp1a and cftr mRNA expression levels in control fish, while in Aroclor 1254-treated fish they remained mostly unchanged. This desensitization also occurred for avtrs in hypothalamus, caudal kidney and liver. In addition, an enhancement in plasma cortisol concentration, together with the orchestration of several players of the Hypothalamic-Pituitary-Interrenal axis (crh, crhbp, trh, star), was also observed mostly at the highest dose used (50 µg g-1 body mass), affecting plasma and hepatic metabolites. Our results demonstrated that Aroclor 1254 compromises the hypoosmoregulatory function of vasotocinergic system in S. aurata, also inducing a concomitant stress response. In summary, this study demonstrates that Aroclor 1254 can be considered an important endocrine disruptor in relation with the correct arrangement of vasotocinergic, metabolic and stress pathways after their stimulation by transfer to hyperosmotic environments.