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A custom microarray was used for the transcriptomic profiling of liver, gills and hypothalamus in response to hypo- (38‰ → 5‰) or hyper- (38‰ → 55‰) osmotic challenges (7 days after salinity transfer) in gilthead sea bream (Sparus aurata) juveniles. The total number of differentially expressed genes was 777. Among them, 341 and 310 were differentially expressed in liver after hypo- and hyper-osmotic challenges, respectively. The magnitude of changes was lower in gills and hypothalamus with around 131 and 160 responsive genes in at least one osmotic stress condition, respectively. Regardless of tissue, a number of genes were equally regulated in either hypo- and hyper-osmotic challenges: 127 out of 524 in liver, 11 out of 131 in gills and 19 out of 160 in hypothalamus. In liver and gills, functional analysis of differentially expressed genes recognized two major clusters of overlapping canonical pathways that were mostly related to “Energy Metabolism” and “Oxidative Stress”. The later cluster was represented in all the analyzed tissues, including the hypothalamus, where differentially expressed genes related to “Cell and tissue architecture” were also over-represented. Overall the response for “Energy Metabolism” was the up-regulation, whereas for oxidative stress-related genes the type of response was highly dependent of tissue. These results support common and different osmoregulatory responses in the three analyzed tissues, helping to load new allostatic conditions or even to return to basal levels after hypo- or hyper-osmotic challenges according to the different physiological role of each tissue. [ABSTRACT FROM AUTHOR]

Abstract: Arginine vasotocin (AVT) and isotocin (IT) levels in plasma and pituitary, and melatonin (MEL) levels in plasma were determined in gilthead sea bream (Sparus auratus) subjected to two different types of stress: i) high density (HD) and ii) food deprivation (NF: non-fed). Fishes were randomly assigned to one of 4 treatments that lasted for 14 days: 1) fed fish under normal low density (ND, 4 kg m−3); 2) non-fed (NF) fish under ND; 3) fed fish under high density (HD, 70 kg m−3); and 4) non-fed fish under HD. Ten fish from each tank were anaesthetized, weighed and plasma and pituitary samples were taken. Plasma and pituitary AVT and IT content were determined by HPLC, while plasma MEL was assayed by RIA. Plasma AVT and IT values were enhanced in all fish kept at high density. The response of AVT was much stronger than that of IT. The highest pituitary AVT and IT levels were shown in NF fish kept at normal density. The significantly higher plasma MEL levels were measured in fed fish kept at HD. These results suggest a role of AVT, IT and MEL in response of sea bream to a common stress factor, high density. Although food deprivation does not influence AVT and IT plasma levels, it seems to affect hypothalamic synthesis of nonapeptides. Further studies are required to elucidate the complex role of AVT, IT and MEL in the sea bream''s response to different stress stimuli. [Copyright &y& Elsevier]

The effect of 17β-estradiol (E2) on osmoregulatory performance was examined in the euryhaline killifish, Fundulus heteroclitus. Fish were injected once with 1, 2 and 5 μg g−1 E2 and, 6 h after injection, transferred from 1 ppt seawater (SW) to full strength SW (40 ppt) or from SW to 1 ppt SW. In another set of experiments, fish were injected four times on alternate days with 2 μg g−1 E2 and then, 6 h after the last injection, transferred from 1 ppt SW to SW or from SW to 1 ppt SW. Fish were sampled 18 h after transfer (i.e., 24 h post-injection), and plasma osmolality, Na+ and Cl− concentration and gill K+-pNPPase activity (a reflection of the sodium pump) were examined. Transfer from 1 ppt SW to SW resulted in significantly increased plasma osmolality, but did not affect gill K+-pNPPase activity. A single dose of E2 (1, 2 and 5 μg g−1) prior to transfer from 1 ppt SW to SW increased plasma osmolality and decreased gill K+-pNPPase activity in a dose-dependent manner. Prolonged treatment with E2 increased plasma osmolality and decreased gill K+-pNPPase activity in 1 ppt SW-adapted fish. Transfer of fish thus treated from 1 ppt SW to SW increased plasma osmolality and did not alter gill K+-pNPPase activity. Transfer from SW to 1 ppt SW had no significant effect on plasma osmolality or gill K+-pNPPase activity. Only the highest single dose of E2 (5 μg g−1) prior to transfer from SW to 1 ppt SW decreased gill K+-pNPPase activity. Prolonged treatment with 2 μg g−1 E2 decreased gill K+-pNPPase activity only following transfer from SW to 1 ppt SW. The results substantiate an inhibitory action of E2 on hypoosmoregulatory capacity in this euryhaline teleost. [Copyright &y& Elsevier]

This work aimed to assess the suitability of a microalgal blend as a dietary ingredient for feeding juveniles of marine carnivorous and herbivorous teleost, as is the case of Sparus aurata and Mugil cephalus, respectively, and to isolate microorganisms from different media and characterize them on the base of their enzymatic activities and their antagonism against important fish pathogens. Thirty juveniles of each species (70 ± 3.2 g S. aurata mean weight and 47 ± 2.8 g M. cephalus mean weight) were distributed in four tanks (15 individuals each) corresponding to four independent dietary treatments (control and microalgae diets designed for each species). Fish were fed their corresponding diets ad libitum for 108 days. At the end of the trial, fish were weighed, and plasma, liver, perivisceral fat, and the entire intestines were obtained for the evaluation of growth performance and metabolic assessment. Furthermore, 117 bacterial strains were isolated in different culture media from the gastrointestinal tract of S. aurata fed the microalgae blend and further characterized for their potential use as probiotics in aquaculture. S. aurata fed the microalgae-supplemented diet (25% dietary inclusion) showed a significant increase in weight gain, specific growth rate, feed efficiency, hepatosomatic, and intestine length indices. However, growth performance and somatic indices in M. cephalus were not affected by the experimental diets. Plasma samples from S. aurata fed the microalgal diet revealed higher levels of glucose and triglycerides and a decrease in cortisol levels. No significant differences were found in any biochemical parameters among the experimental diets in M. cephalus. In conclusion, both species demonstrated a favorable adaptation to the nutritional formulation employed in this study, and bacterial strains UMA-169 and UMA-216 (both identified as Bacillus pumilus) could be considered for use in aquaculture as they might benefit host health by improving digestion and absorption of different energy sources and by minimizing the colonization of pathogenic species. [ABSTRACT FROM AUTHOR]

Simple Summary: The aquaculture industry is constantly looking for alternative ingredients to fishmeal and terrestrial-plant feedstuffs. Here, we have studied the effects of the combined administration of Chlorella fusca and ethanol-inactivated Vibrio proteolyticus DCF12.2 in the diet of the thick-lipped grey mullet (Chelon labrosus), a promising candidate species for aquaculture diversification. The combined diet enhanced growth performance, n-3 polyunsaturated fatty acids, and fish lipid quality. Furthermore, the experimental diet increased several metabolic enzymes' activity, as well as the intestinal absorption capacity. It can thus be concluded that the experimental diet composed of C. fusca and V. proteolyticus was suitable for feeding C. labrosus. 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. [ABSTRACT FROM AUTHOR]

Activities of pepsin, trypsin, chymotrypsin, alpha-amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick-lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS-PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha-amylase and no lipase activity recorded in pre-adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit. [ABSTRACT FROM AUTHOR]

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 (~ 3 min) 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 1 h), 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. [ABSTRACT FROM AUTHOR]

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 Aqpl paralog gene function in the teleost gilthead sea bream (Sparus aurata). The complete coding regions of Aqpla, Aqplb, 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 Aqplb mRNA revealed regulation of water transport via PKA (IBMX+forskolin sensitive), whereas Aqpla mRNA injection had the same effect via PKC signaling (PDBU sensitive). In the absence of expressed receptors, AVT and IT (10-8 mol I-1) were unable to modify AQP1 function. AVT regulated AQP1a and AQP1b function only when the AVTR V2-type was coexpressed. IT regulated AQPIafunction, but not AQP1b, only when ITR was present. Considering that Aqpla and Aqplb 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. [ABSTRACT FROM AUTHOR]

Simple Summary: It is generally accepted (in mammals and in teleost fish, too) that stressful conditions affect the performance of an immune response. What is still far from being known is at what extend does an immune process affects the neuroendocrine system. Vaccination for instance, is nowadays a common practice in aquaculture and little is known about its physiological implications other than immunization. Here is a first approach to the study of the European seabass' brain gene expression patterns in response to a peripheral inflammatory process. Genes related to the stress response were focused, along with those related to the opioid system. Increased expression of certain genes suggests the activation of a stress response triggered by inflammatory signals. Additionally, contrasting expression patterns of the same gene (increased vs decreased) in the different brain regions (as well as the time needed for changes to happen) point at different functions. These results clearly show the reactivity of different brain responses to an immune response, highlighting the importance of further studies on downstream implications (behavior, feeding, welfare, reproduction). In fish, as observed in mammals, any stressful event affects the immune system to a larger or shorter extent. The neuroendocrine-immune axis is a bi-directional network of mobile compounds and their receptors that are shared between both systems (neuroendocrine and immune) and that regulate their respective responses. However, how and to what extent immunity modulates the neuroendocrine system is not yet fully elucidated. This study was carried out to understand better central gene expression response patterns in a high-valued farmed fish species to an acute peripheral inflammation, focusing on genes related to the hypothalamus-pituitary-interrenal axis and the opioid system. European seabass, Dicentrarchus labrax, were intra-peritoneally injected with either Freund's Incomplete Adjuvant to induce a local inflammatory response or Hanks Balances Salt Solution to serve as the control. An undisturbed group was also included to take into account the effects due to handling procedures. To evaluate the outcomes of an acute immune response, fish were sampled at 4, 24, 48, and 72 h post-injection. The brain was sampled and dissected for isolation of different regions: telencephalon, optic tectum, hypothalamus, and pituitary gland. The expression of several genes related to the neuroendocrine response was measured by real-time PCR. Data were statistically analyzed by ANOVA and discriminant analyses to obtain these genes' responsiveness for the different brain regions. Serotonergic receptors were upregulated in the telencephalon, whereas the optic tectum inhibited these transcription genes. The hypothalamus showed a somewhat delayed response in which serotonin and glucocorticoid receptors were concerned. Still, the hypothalamic corticotropin-releasing hormone played an important role in differentiating fish undergoing an inflammatory response from those not under such conditions. Opioid receptors gene expression increased in both the hypothalamus and the telencephalon, while in the optic tectum, most were downregulated. However, no changes in the pituitary gland were observed. The different brain regions under immune stimulation demonstrated clear, distinct responses regarding gene transcription rates as well as the time period needed for the effect to occur. Further, more integrative studies are required to associate functions to the evaluated genes more safely and better understand the triggering mechanisms. [ABSTRACT FROM AUTHOR]

Aquaculture is considered one of the most important food production systems both in terms of economic impact and food security, and the ongoing development of this industry is a key factor in the strategy to guarantee global nutritional safety. Nowadays, different types of nanotechnology-based systems have been employed to increase its production, efficiency and sustainability. Recent efforts have been made in the fields of health management, enhancement of fish and shellfish development by dietary supplementation with nutraceuticals, but also in the processing and preservation of seafood and water treatment, among others. Therefore, nanotechnology has a significant role to play in the improvement of the efficiency and the environmental impact of this industry. Given this perspective, we propose to review the current situation of nanotechnology in the field of aquaculture and fisheries, emphasizing not only in current applications, and future prospects, but also in the ethical and governance aspects associated with this topic. [ABSTRACT FROM AUTHOR]

Simple Summary: Decapod crustaceans live in practically all marine, freshwater, and semi-terrestrial habitats on Earth, and exhibit a remarkable variation in their feeding behavior, from filter feeding, grazing, and scavenging to hunting. However, most knowledge about digestive biochemistry in crustaceans has come from studies on a few economically relevant species due to the importance of optimized formulated feeds for aquaculture success. Moreover, most data on α-amylases in decapods derived from studies in herbivore and omnivore species. There are few reviews addressing different aspects of the digestive physiology of decapods, including data on digestive enzymes, but no comprehensive review is available on α-amylases in this group and, in general, information on carnivorous species is often neglected. This review summarizes the information obtained on decapods' α-amylases and uses recent data from a carnivorous lobster as a connecting thread to compare features of α-amylases from species with different feeding habits, drawing a more comprehensive view of the role of α-amylases across decapods crustaceans. Decapod crustaceans are a very diverse group and have evolved to suit a wide variety of diets. Alpha-amylases enzymes, responsible for starch and glycogen digestion, have been more thoroughly studied in herbivore and omnivore than in carnivorous species. We used information on the α-amylase of a carnivorous lobster as a connecting thread to provide a more comprehensive view of α-amylases across decapods crustaceans. Omnivorous crustaceans such as shrimps, crabs, and crayfish present relatively high amylase activity with respect to carnivorous crustaceans. Yet, contradictory results have been obtained and relatively high activity in some carnivores has been suggested to be a remnant trait from ancestor species. Here, we provided information sustaining that high enzyme sequence and overall architecture conservation do not allow high changes in activity, and that differences among species may be more related to number of genes and isoforms, as well as transcriptional and secretion regulation. However, recent evolutionary analyses revealed that positive selection might have also occurred among distant lineages with feeding habits as a selection force. Some biochemical features of decapod α-amylases can be related with habitat or gut conditions, while less clear patterns are observed for other enzyme properties. Likewise, while molt cycle variations in α-amylase activity are rather similar among species, clear relationships between activity and diet shifts through development cannot be always observed. Regarding the adaptation of α-amylase to diet, juveniles seem to exhibit more flexibility than larvae, and it has been described variation in α-amylase activity or number of isoforms due to the source of carbohydrate and its level in diets, especially in omnivore species. In the carnivorous lobster, however, no influence of the type of carbohydrate could be observed. Moreover, lobsters were not able to fine-regulate α-amylase gene expression in spite of large changes in carbohydrate content of diet, while retaining some capacity to adapt α-amylase activity to very low carbohydrate content in the diets. In this review, we raised arguments for the need of more studies on the α-amylases of less studied decapods groups, including carnivorous species which rely more on dietary protein and lipids, to broaden our view of α-amylase in decapods crustaceans. [ABSTRACT FROM AUTHOR]

Fish held at high stocking densities are generally exposed to chronic stress situations that impose severe energy demands and may predispose the fish to infection. Senegalese sole Solea senegalensis (Kaup) juveniles (78.8±18.9 g body weight) were maintained at low (LSD; 4 kg m−2 at the end of the experiment), medium (MSD; 9 kg m−2) and high (HSD; 14 kg m−2) stocking densities during a period of 63 days. Although disease outbreaks were observed in fish reared at HSD, growth and food consumption did not vary among different treatments. Results from plasma cortisol and free amino acids (FAAs) showed significant differences among different rearing densities pointing to HSD as stressful rearing condition. However, higher plasma glucose and osmolality levels indicated that fish held at MSD may also be under stress. The higher usage of FAAs from the HSD group may be due to the higher demand for energy production in order to cope with stressful rearing conditions, higher rate of protein synthesis or due to synthesis of other important metabolites related to stress response. Therefore, results from the present study point out to HSD as a stressful rearing condition and suggest that crowding stress may affect amino acid requirements. [ABSTRACT FROM AUTHOR]

Given the potential of algae as a new aquafeed ingredient, this study evaluated the effect of the tank‐cultivated macroalgae Ulva rigida C. Agardh (Chlorophyta) on innate immune response, intestinal microbiota and their resistance against Photobacterium damselae subsp. piscicida in juvenile gilthead seabream (Sparus aurata L.) after a short pulse of administration. An algae‐free diet was used as control, and an experimental diet was formulated containing 25% of U. rigida (UL‐25). After 2, 7, 15 and 30 days biological samples were taken. No statistical significance (p > 0.05) was detected in respiratory burst activity. Diet UL‐25 significantly enhanced alternative complement pathway activity respect to control diet from the beginning of the experiment (p < 0.05). When fish were challenged against P. damselae subsp. piscicida, UL‐25‐fed fish were more resistant against the pathogen than the fish fed control diet, obtaining a relative survival percentage of 54.55%. The ability of UL‐25 diet to modify the intestinal microbiota was also confirmed. In conclusion, the use of a short pulse of dietary administration of 25% U. rigida in S. aurata diet modulates the immune response, intestinal microbiota and reduced the mortality rate during P. damselae subsp. piscicida infection. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate the effects of the dietary inclusion of Arthrospira sp. enzyme hydrolysate on gilthead seabream (Sparus aurata) juveniles in a 128-day feeding trial. Algal hydrolysate was tested at low inclusion level, namely, 2 and 4%, against a control diet without Arthrospira sp. supplementation. At the end of the feeding trial, fish body weight was recorded for growth evaluation. No significant differences were found among the experimental groups regarding growth performance or nutrient utilization, despite the fact that those animals fed with diets enriched with Arthrospira displayed higher trypsin, chymotrypsin, and leucine aminopeptidase enzyme activities, compared to fish fed with control diet. The ultrastructural study of the intestinal mucosa also revealed increased microvilli length and absorptive capacity in fish fed with Arthrospira sp. diets, especially at 4% inclusion level. Arthrospira supplementation was also responsible for lower lipid oxidation in muscle tissue, as well as for remarkable colour differences in skin, compared to control animals. These results suggest, overall, that low dietary supplementation with this microalgal hydrolysate might improve not only the intestinal ultrastructure and functionality but also muscle pigmentation and antioxidant capacity of juvenile gilthead seabream. [ABSTRACT FROM AUTHOR]

• Aroclor 1254 exposure influences endocrine and osmoregulatory systems in fish acclimated to SW. • The energetic reorganization due to the stress situation induced in HSW-transferred fish is exacerbated by Aroclor 1254. • A clear blockage of vasotocinergic system is produced by Aroclor 1254 at central, plasma and peripheral levels. • Aroclor 1254 compromises the hypoosmoregulatory function of vasotocinergic system. 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 avtr s 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. [ABSTRACT FROM AUTHOR]

• Silver catfish physiology may be affected by the interaction of feeding and stocking density. • The expression of several genes related to the stress cascade is influenced by both factors. • Low stocking density impairs the physiological status of silver catfish. • Fish maintained in high stocking density showed higher responsiveness to fasting. 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. [ABSTRACT FROM AUTHOR]

Studies on molecular characteristics and modulation of expression of α-amylase and trypsin in the hepatopancreas of intertidal euryhaline crabs are lacking. In this work, we cloned and studied by in silico approaches the characteristics of cDNA sequences for α-amylase and two trypsins isoforms, as well as the effect of environmental salinity, on gene expression and protein activities in the hepatopancreas of Neohelice granulata (Dana, 1851), which is a good invertebrate model species. The cDNA sequence of α-amylase is 1637 bp long, encoding 459 amino acid residues. Trypsin 1 and 2 are 689 and 1174 bp long, encoding 204 and 151 amino acid residues, respectively. Multiple sequence alignment of deduced protein sequences revealed the presence of conserved motifs found in other invertebrates. In crabs acclimated at 37 psu (hyporegulation), α-amylase mRNA level and total pancreatic amylase activity were higher than at 10 psu (hyperregulation) and 35 psu (osmoconformation). Trypsin 1 mRNA levels increased at 37 psu, while trypsin 2 levels decreased at 10 and 37 psu. Total trypsin activity was similar in all salinities. Our results showed a differential modulation of α-amylase and trypsin expression and total amylase activity by salinity acclimation, suggesting the occurrence of distinct mechanisms of regulation at different levels that could lead to digestive adjustments in relation to hyperregulation and (or) hyporegulation. [ABSTRACT FROM AUTHOR]

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; 5 kg·m − 3 ); (2) fed at high stocking density (HSD-F, 40 kg·m − 3 ); (3) food-deprived at LSD (LSD-FD); and (4) food-deprived at HSD (HSD-FD). After 21 days, 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 . [ABSTRACT FROM AUTHOR]

In keeping with the premises of Blue Circular Economy in the European Union, the present study explored the possibility of using the invasive brown alga Rugulopteryx okamurae in aquafeeds for European sea bass (Dicentrarchus labrax). Assuming the raw algae biomass could negatively impact animal performance, four experimental formulations were prepared, by including macroalgae material at 5%, using crude (CR), enzymatically hydrolysed and fermented (EF), enzymatically hydrolysed (E), or fermented (F) R. okamurae biomass, which we tested against a control feed (CT). To evaluate the effects of the experimental diets, besides animal growth performance and biometric parameters, we devised a toolbox focused on the intestine and intestinal function: i) ex-vivo epithelial resistance and permeability in Ussing chambers; ii) microbiota composition through NGS; iii) expression profiles of selected markers for epithelial integrity, transport, metabolism, and immune response, by qPCR. Our results show differentiated allometric growth among diets, coupled with intestinal epithelium alterations in permeability, integrity, and amino acid transport. Additionally, evidence of microbiota dysbiosis and contrasting immune responses between experimental diets, i.e. pro-inflammatory vs. anti-inflammatory, are also described. In conclusion, we believe that R. okamurae could be a suitable resource for aquafeeds for the European sea bass, although its use requires a pre-treatment before inclusion. Otherwise, while the fish still have a positive growth performance, the gastrointestinal tract pays a toll on the integrity, transport, and inflammatory processes. • Rugulopteryx okamurae is an invasive brown alga species in southern Europe. • Sustainable strategies for using the accumulating algal biomass need to be tested. • Adding raw R. okamurae to aquafeeds affects intestinal processes in European seabass. • Algal biomass enzymatic hydrolysis/fermentation pre-treatments significantly reduce impact. [ABSTRACT FROM AUTHOR]

This study aimed to determine the maximum time of emersion of Senegalese sole ( Solea senegalensis) specimens to form the basis for a dry sole transport procedure. For this purpose, 56 specimens (922.33 ± 29.52 g body weight) were individually placed in polystyrene boxes on a wet cloth, maintaining constant temperature (15°C) during emersion trial. Specimens ( n = 7 per group) were sampled for plasma, and mortality was monitored at different points (0, 2, 6, 12, 20, 28, 36 and 44 h of emersion), and several stress (cortisol, glucose and lactate) and metabolic (triglycerides, proteins and amino acids) parameters were assessed. After sampling, each experimental group was placed in recovery tanks for the assessment of delayed mortality and feeding performance after emersion trial. Transport periods longer than 28 h produced high stress level indicated by (i) significant increase in circulating cortisol and lactate and (ii) glucose reserves mobilization. These alterations originated physiological problems during the emersion trial that induced mortality as well as problems for recovering correct feeding performance of specimens. These results suggested that at 15°C for S. senegalensis specimens (around 900 g body weight), a waterless transport is possible without direct or delayed mortality up to 28 h. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the inclusion of two macroalgae as dietary ingredients on growth, body composition, intermediate metabolism, intestinal enzymes and gut histology of Sparus aurata juveniles. Six isocaloric and isonitrogenous diets incorporating 5%, 15% or 25% of Gracilaria cornea or Ulva rigida meal were tested against a control diet. Duplicate groups of fifteen fish (14 g) were fed the experimental diets for 70 days. Growth performance showed an inverse linear relationship with the inclusion level of Gracilaria. Feed conversion ratio increased significantly in fish fed 25% Gracilaria. Ulva presented a quadratic relationship with the lowest SGR at 15% of inclusion. Fish fed 25% Ulva exhibited significantly lower lipid content than those fed control diet. Overall, metabolites did not show differences with specimens fed control diet, except for plasmatic glucose and muscle lactate. Intestinal proteolytic activity was reduced with the macroalgae inclusion level, but all fish showed the same enzyme pattern. The histological study revealed no negative effect on liver and intestine structure by the macroalgae inclusion. The present work demonstrated that these ingredients can be used in practical diets for sea bream, but a more comprehensive research is needed to determine the optimum dietary level in a longer feeding trial. [ABSTRACT FROM AUTHOR]

• Sparus aurata were exposed to environmental concentrations of CIP, TRIM and SULF. • Exposure to CIP and TRIM produced oxidative stress in the liver. • CIP altered the expression of genes related to the HPI and HPT axes in the brain. • Most molecular alterations were produced after CIP exposure. • Gene expression was not significantly altered after SULF and TRIM treatments. The high consumption and subsequent input of antibacterial compounds in marine ecosystems has become a worldwide problem. Their continuous presence in these ecosystems allows a direct interaction with aquatic organisms and can cause negative effects over time. The objective of the present study was to evaluate the effects of exposure to three antibacterial compounds of high consumption and presence in marine ecosystems (Ciprofloxacin CIP, Sulfadiazine SULF and Trimethoprim TRIM) on the physiology of the gilthead sea bream, Sparus aurata. Plasma parameters, enzymatic biomarkers of oxidative stress and damage and expression of genes related to stress and growth were assessed in exposed S. aurata specimens. For this purpose, sea bream specimens were exposed to individual compounds at concentrations of 5.2 ± 2.1 μg L −1 for CIP, 3.8 ± 2.7 μg L −1 for SULF and 25.7 ± 10.8 μg L −1 for TRIM during 21 days. Exposure to CIP up-regulated transcription of genes associated with the hypothalamic-pituitary-thyroid (HPT) (thyrotropin-releasing hormone, trh) and hypothalamic-pituitary-interrenal (HPI) axes (corticotropin-releasing hormone-binding protein, crhbp) in the brain, as well as altering several hepatic stress biomarkers (catalase, CAT; glutathione reductase, GR; and lipid peroxidation, LPO). Similar alterations at the hepatic level were observed after exposure to TRIM. Overall, our study indicates that S. aurata is vulnerable to environmentally relevant concentrations of CIP and TRIM and that their exposure could lead to a stress situation, altering the activity of antioxidant defense mechanisms as well as the activity of HPT and HPI axes. [Display omitted] [ABSTRACT FROM AUTHOR]

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase. [ABSTRACT FROM AUTHOR]

The influence of culture density on growth, stress system and metabolism has been assessed in A. regius juveniles submitted to different initial stocking densities (3, 7, 10 and 13 g L − 1 ) for 40 days. On days 0, 12, 27 and 40 of the experiment, biometric parameters were measured. In addition, plasma, liver and muscle samples were taken for biochemical analysis at the end of experimental time. Our results showed better growth rates and feed utilization in those specimens held at high stocking densities when compared to those under low stocking densities. In addition, higher plasma cortisol, glucose and triglycerides levels found in the lowest stocking density tested suggest an activation of the stress system in fish held at this condition. Moreover, no changes in hepatic metabolites were observed, while values of muscle triglycerides, amino acids and lactate enhanced when stocking density increased. In conclusion, the culture of juvenile A. regius at higher initial densities (at least 13 g L − 1 ) ensures better growth and the optimization of the existing facilities, improving welfare of this species in aquaculture systems. [ABSTRACT FROM AUTHOR]

Fish are usually exposed to farm operations that can activate the stress system, which in turn diverts energy from normal metabolic processes (i.e. growth or immune defence) to deliver it into the physiological systems activated to cope with stress. The main goal of this study was to assess the effect of high stocking densities on growth performance of Senegalese sole ( Solea senegalensis ) juveniles reared under intensive farming conditions. The impact of high densities on plasma cortisol and metabolites, humoral immune parameters and hepatic oxidative status was evaluated. Senegalese sole juveniles (45 ± 5 g average body weight) were stocked at three different initial densities (7, 17 and 24 kg m − 2 ) throughout a 60 days rearing period. Final stocking densities were 13, 31 and 40 kg m − 2 with no differences in terms of growth performance. Similar levels of plasma cortisol, circulating leucocytes and metabolites and humoral innate immune parameters suggest that stocking densities do not affect the hematological and biochemical status of Senegalese sole juveniles, at least under the current experimental conditions. Overall, this study presents the possibility for Senegalese sole on-growing at high stocking densities, strengthening the needs for its intensive commercial farming. [ABSTRACT FROM AUTHOR]

The influence of stocking density and food deprivation on energy metabolism, stress processes and the pituitary endocrine system of silver catfish ( Rhamdia quelen ) was investigated after a period of 14 days, in which plasmatic and hepatic parameters and the mRNA expression of prolactin (PRL), growth hormone (GH) and somatolactin (SL) were assessed. The fish were subjected to four experimental conditions: (1) fed under high stocking density (32 kg/m 3 , HSD); (2) fed under mean stocking density (16 kg/m 3 , MSD); (3) fed under low stocking density (8 kg/m 3 , LSD); and (4) food-deprived under low stocking density (8 kg/m 3 , LSD-FD). After 14 days, plasma and liver samples were obtained to analyze the metabolite levels and enzymatic activities related to metabolism, and pituitary glands were obtained to analyze hormone expression (PRL, GH and SL). Liver weight and the hepato-somatic index (HSI) revealed that specimens maintained at HSD and/or MSD had higher hepatic stores, which were observed in the triglyceride and glycogen levels in this tissue, than animals submitted to the LSD and LSD-FD groups. Triglyceride levels in the plasma and liver revealed the consumption of fatty acid reserves in the fasting group. Enzymatic activities, such as glutamate dehydrogenase (GDH), phosphorylase (GPase), pyruvate kinase (PK), aspartate transaminase (AST) and glycerol-3-phosphate dehydrogenase (G3PDH), indicated an increase in gluconeogenic pathways in the HSD group and an increase in glycolitic metabolism in the LSD groups. The expression of PRL was not affected by stocking density and/or food deprivation and GH decreased with increased density and increased in fasting conditions. A negative effect of density and fasting was observed on the expression of SL. Overall, the data suggested that juvenile silver catfish reared at stocking densities of 16 to 32 kg/m 3 were better maintained than those maintained at the lowest density. [ABSTRACT FROM AUTHOR]

In this study, the protective effects of diphenyl diselenide [(PhSe)2] on quinclorac- induced toxicity were investigated in silver catfish (Rhamdia quelen). The fish were fed for 60 days with a diet in the absence or in the presence of 3.0 mg/Kg (PhSe)2. Animals were further exposed to 1 mg/L quinclorac for 8 days. At the end of experimental period, fish were euthanized and biopsies from liver and gills, as well as blood samples, were collected. The cortisol and metabolic parameters were determined in plasma, and those enzyme activities related to osmoregulation were assayed in the gills. In liver, some important enzyme activities of the intermediary metabolism and oxidative stress-related parameters, such as thiobarbituric acid-reactive substance (TBARS), protein carbonyl, catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), nonprotein thiols (NPSH) and ascorbic acid contents were also evaluated. Compared to the control group, quinclorac exposure significantly decreased hepatosomatic index and increased cortisol and lactate values in plasma. Moreover, the activities of fructose biphosphatase (FBPase), glucose-6-phosphate dehydrogenase (G6Pase), glycogen phosphorilase (GPase) and aspartate aminotransferase (AST) were significantly increased in liver. Quinclorac also induced lipid peroxidation while the activity of SOD, NPSH and ascorbic acid levels decreased in the liver. However, dietary (PhSe)2 reduced the herbicide-induced effects on the studied parameters. In conclusion, (PhSe)2 has beneficial properties based on its ability to attenuate toxicity induced by quinclorac by regulating energy metabolism and oxidative stress-related parameters. [ABSTRACT FROM AUTHOR]

Since fish show daily rhythms in most physiological functions, it should not be surprising that stressors may have different effects depending on the timing of exposure. In this study, we investigated the influence of time of day on the stress responses, at both physiological and cellular levels, in gilthead sea bream ( Sparus aurata L.) submitted to air exposure for 30 s and then returned to their tank. One hour after air exposure, blood, hypothalamus and liver samples were taken. Six fish per experimental group (control and stressed) were sampled every 4 h during a 24-h cycle. Fish were fed in the middle of the light cycle (ML) and locomotor activity rhythms were recorded using infrared photocells to determine their daily activity pattern of behaviour, which showed a peak around feeding time in all fish. In the control group, cortisol levels did not show daily rhythmicity, whereas in the stressed fish, a daily rhythm of plasma cortisol was observed, being the average values higher than in the control group, with increased differences during the dark phase. Blood glucose showed daily rhythmicity in the control group but not in the stressed one which also showed higher values at all sampling points. In the hypothalamus of control fish, a daily rhythm of corticotropin-releasing hormone ( crh) gene expression was observed, with the acrophase at the beginning of the light phase. However, in the stressed fish, this rhythm was abolished. The expression of crh-binding protein ( crhbp) showed a peak at the end of the dark phase in the control group, whereas in the stressed sea bream, this peak was found at ML. Regarding hepatic gene expression of oxidative stress biomarkers: (i) cytochrome c oxidase 4 showed daily rhythmicity in both control and stressed fish, with the acrophases located around ML, (ii) peroxiredoxin ( prdx) 3 and 5 ( prdx5) only presented daily rhythmicity of expression in the stressed fish, with the acrophase located at the beginning of the light cycle and (iii) uncoupling protein 1 showed significant differences between sampling points only in the control group, with significantly higher expression at the beginning of the dark phase. Taken together, these results indicate that stress response in gilthead sea bream is time-dependent as cortisol level rose higher at night, and that different rhythmic mechanisms interplay in the control of neuroendocrine and cellular stress responses. [ABSTRACT FROM AUTHOR]

Gilthead seabream is a fish species of great importance in Mediterranean aquaculture, attracting many studies on nutrition and chronobiology, although nothing is known about the effect of feeding frequency on the daily rhythms of the gastric digestion process. In this article, we investigated daily rhythms in stomach fullness, gastric and intestine pH, as well as pepsin activity and expression of pepsinogen and proton pump in juvenile fish under three different feeding protocols: (A) one daily meal at 9:00, (B) two daily meals at 9:00 and 17:00 and (C) continuous feeding during the daytime. The results revealed that feeding protocol affected significantly the rhythm of gastric pH and the pepsin activity pattern. The gastric pH exhibited significant daily rhythms in the three cases with the acrophase located at night in the regimes A and B and during daytime, in the regime C. In the regimes A and B, the pepsin activity peaked few hours after the meals, although the afternoon meal in B produced a higher peak. In the regime C, the peak occurred in the middle of the feeding period. Lowest total pepsin activity was observed in regime A, and the highest activity with the regime C. In contrast, the pepsinogen gene expression remained low along the daily cycle, with an expression peak just before or after the morning meal in regimes A and C, respectively. The proton pump gene expression was also practically constant with a peak right after the morning meal in the regime C. On the other hand, intestinal pH showed a postprandial increase after the first morning meal in all the three treatments, recovering the resting values in the dark period. Two meals and continuous feeding allowed a better and prolonged gastric digestion and consequently the juveniles exhibited better growth with the same daily ration of food. In short, while the gastric digestion pattern is mainly driven by pH changes induced by the time of food ingestion, the regulation of the intestinal digestion seems to be more independent of the feeding protocol. [ABSTRACT FROM AUTHOR]

Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2–6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6–12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients. [ABSTRACT FROM AUTHOR]

The aquaculture sector has experienced rapid and important growth with the subsequent increase of feeding and nutritional issues for sustaining this activity, mainly related to the use of high quality, safe and environmentally friendly feed ingredients. The use of additives in aquafeeds has proven to be a suitable option to improve different productive indicators in farmed fish. In the present study, the effect of adding the GHRP-6 peptide, a ghrelin analog, to a commercial diet of gilthead sea bream (Sparus aurata) was studied at two proportions (100 or 500 μg/kg of feed). Both experimental diets show an increase in growth performance, as well as in feed efficiency after 97 days of experiment. The lower inclusion of GHRP-6 (100 μg/kg) results in a better aerobic metabolism, while the higher inclusion significantly increased plasma GH levels in agreement with the GH secretagogue effects of ghrelin. Similar growth outcome and differences between GHRP-6 levels in aerobic metabolism and GH stimulation suggest that improvements in culture performance by this peptide may occur through different mechanisms. Taken together, this compound can be considered as a viable dietary supplement for increasing production efficiency of sea bream aquaculture, although a better understanding of its dose-specific effects is still required. [ABSTRACT FROM AUTHOR]

Highlights: [•] Fish were subjected to three NH3 levels and two hardness levels for 1 and 5 days. [•] Full-length PRL and SL as well as β-actin cDNAs from R. quelen were cloned. [•] Plasma cortisol, metabolites, ions and expression of GH, PRL and SL were determined. [•] High hardness improved endocrine parameters evaluated in catfish at high NH3 levels. [Copyright &y& Elsevier]

Highlights: [•] A partial sequence of AVT V1a2-type receptor was cloned in the sea bream. [•] The AVT V1a2-type receptor is distributed in the whole gastrointestinal tract. [•] The AVT V1a2-type receptor expression is sensitive to salinity. [•] AVT controls ion transport in the intestine as demonstrated in Ussing chambers. [•] AVT action is mediated by a bumetanide-sensitive mechanism, likely NKCC2. [ABSTRACT FROM AUTHOR]

Evaluating the survival of discarded species is gaining momentum after the new European Common Fisheries Policy (Article 15 of the European Regulation No. 1380/2013). This regulation introduced a discard ban, with an exemption for those species with demonstrated high survival rates after their capture and release. Candidate species should be evaluated for each fishing gear and geographical area. In this study, we assessed not only survival, but also physiological recovery rates of blackspot seabream (Pagellus bogaraveo) below commercial size captured with a hookline called "voracera" in the Strait of Gibraltar (SW Atlantic waters of Europe). Experiments onboard a commercial fishing vessel were paralleled with studies in controlled ground-based facilities, where the capture process was mimicked, and physiological recovery markers were described. Our results confirmed that hookline capture induced acute stress responses in the target species, such as changes in plasma cortisol, lactate, glucose, and osmolality. However, 90.6% of the blackspot seabreams below commercial size captured with this fishing gear managed to survive, and evidenced physiological recovery responses 5 h after capture, with complete homeostatic recovery occurring within the first 24 h. Based on this study, the European Commission approved an exemption from the discard (EU Commission Delegated Regulation 6794/2018). Thus, the robust methodology described herein can be an important tool to mitigate the problem of discards in Europe. [ABSTRACT FROM AUTHOR]

Aquaculture procedures usually induce stress that affects the physiological status of fish. For this reason, the inclusion of additives in fish feeds to palliate stress might be a good alternative. This study aimed to assess the antimicrobial activity of citronella (Cymbopogon nardus) essential oil (CEO) against bacterial pathogens and to determine its dietary impact on the growth performance of Sparus aurata. In vitro tests confirmed that CEO possesses antimicrobial activity against several fish-specific pathogens. For the in vivo tests, three experimental groups were fed for 60 days with different concentrations of CEO: CTRL (0 mL kg−1 fish feed); CEO1 (1 mL kg−1 fish feed); and CEO2 (2 mL kg−1 fish feed). At the end of the experiment, the physiological status was characterized. Subsequently, the specimens of the CTRL and CEO2 groups were subjected to a challenge with an injection of Poly I:C for immune stimulation. Although S. aurata individuals tolerated CEO inclusion without compromising growth performance, it significantly reduced glycogen in the CEO2 group, concomitant to an increment of total peripheral leucocytes. Moreover, different hematological profiles' responsive patterns against an inflammatory stimulus were observed. In conclusion, our results suggest that the use of CEO as a fish feed additive can prevent bacterial outbreaks and improve potential in vivo disease resistance in S. aurata without negatively affecting growth. [ABSTRACT FROM AUTHOR]

Simple Summary: Cortisol is a key stress hormone in teleosts. Cortisol exerts its effects through genomic—and membrane-initiated mechanisms, however, the role of the latter in long-term stress responses is unknown. Here, we treated Sparus aurata with cortisol or cortisol-BSA (exclusive inductor to membrane-initiated effects) to emulate a long-term stress situation. We found that cortisol, but not cortisol-BSA, promotes energy substrate mobilization in the liver, together with the regulation of metabolism-related genes. We suggest that genomic cortisol actions exclusively participate in metabolic responses during prolonged treatment using cortisol in S. aurata. This study contributes to the current knowledge on cortisol's involvement in stress responses in fish. Cortisol is the main glucocorticoid hormone promoting compensatory metabolic responses of stress in teleosts. This hormone acts through genomic and membrane-initiated actions to exert its functions inside the cell. Experimental approaches, using exogenous cortisol administration, confirm the role of this hormone during short (minutes to hours)- and long-term (days to weeks) responses to stress. The role of membrane-initiated cortisol signaling during long-term responses has been recently explored. In this study, Sparus aurata were intraperitoneally injected with coconut oil alone or coconut oil containing cortisol, cortisol-BSA, or BSA. After 3 days of treatment, plasma, liver, and skeletal muscle were extracted. Plasma cortisol, as well as metabolic indicators in the plasma and tissues collected, and metabolism-related gene expression, were measured. Our results showed that artificially increased plasma cortisol levels in S. aurata enhanced plasma glucose and triacylglycerols values as well as hepatic substrate energy mobilization. Additionally, cortisol stimulated hepatic carbohydrates metabolism, as seen by the increased expression of metabolism-related genes. All of these responses, observed in cortisol-administered fish, were not detected by replicating the same protocol and instead using cortisol-BSA, which exclusively induces membrane-initiated effects. Therefore, we suggest that after three days of cortisol administration, only genomic actions are involved in the metabolic responses in S. aurata. [ABSTRACT FROM AUTHOR]

Abstract: The mobilization of nutrients and sexual hormone changes during the reproductive cycle of the Mediterranean mussel (Mytilus galloprovincialis), as well as sex related differences have been assessed using adults brought from the field. Metabolites (glucose and glycogen) and lipid composition were determined in the mantle and digestive gland. Moreover, glucose and aminoacids in hemolymph have also been measured. Glucose and glycogen values were lower in ripeness stage in both sexes and tissues analyzed as well as glucose in hemolymph. Glucose was mobilized during sexual maturation to be accumulated as glycogen, which was used as an energy source explaining the low levels in ripeness stage. Lipid content enhanced with ripening in the female mantle while in the digestive gland increased at the beginning but decreased at the end in both sexes. These results suggested the accumulation of lipid for larval development in eggs as well as the energy source during gametogenesis. Triglycerides and phospholipids were the major lipid components in both sexes and tissues assessed, showing several changes mainly in males. Increasing levels of n-3 polyunsaturated fatty acids (PUFA) during gonad maturation mainly due to 22:6n-3 (DHA), have been also observed. Total lipids were higher in females than in males, while triglicerides dominated in females and phospholipids in males. The main fatty acids in both sexes were 16:0, 20:5n-3 (EPA) and DHA. Females showed higher percentages of monounsaturated fatty acids (MUFA) and lower level of saturated fatty acids (SAFA) than males. Sexual hormones have also been analyzed and changes in progesterone and 17-β estradiol during gonadal development have been detected in both sexes. These data will help hatchery managers to determine the best nutritive strategy for producing large quantities of mussel seed. [Copyright &y& Elsevier]

In this work, the wedge sole ( Dicologoglossa cuneata) spawning under a natural thermocycle has been studied. The spawning parameters were similar to those of other flatfish in captivity, the relative fecundity being very high (1.7 ± 0.1 × 10 eggs kg female per spawning season). The percentage of floating eggs decreased with time and was directly related to egg diameter, daily relative fecundity and fertilization rate. Nevertheless, the hatching rate variations were not correlated to any other spawning parameter. The temperature range during the spawning season was very wide (11-21°C) though the largest spawns occurred between 12 and 16°C. The natural temperature cycle influenced wedge sole egg production, the winter temperature minimum indicating the onset of spawning, but the variations in the reproductive season did not appear to affect the daily egg production. Future research should focus on the photoperiod and its interaction with temperature as key factor for spawning. [ABSTRACT FROM AUTHOR]

The effects of two different environmental salinities [brackish water (BW), 12‰; sea water (SW), 39‰] and initial stock densities [low (LD), 1.0 g L−1; high (HD), 2.0 g L−1] on growth, osmoregulation, stress and energy metabolism of the fry Pagrus pagrus were investigated over a period of 45 days. Pagrus pagrus ( n=80, 5.51 ± 0.25 g mean initial body weight) were randomly divided in eight groups. Growth, weight gain and specific growth rate increased in BW-acclimated fish compared with SW-acclimated fish. No differences were observed between the two stock densities tested at either environmental salinity. Plasma osmolality was lowest in BW-acclimated specimens, but the stock density had no effect on this parameter. Branchial Na+,K+-ATPase activity was positively correlated with environmental salinity, but unaltered at the renal level. Plasmatic parameters were enhanced by salinity and stocking conditions. At the hepatic level, triglyceride values were enhanced in BW-acclimated fish maintained at LD. Muscle metabolites (glycogen, glucose and lactate) increased in BW- compared with SW-acclimated fish; stock density had no influence. Our data suggest that changes in metabolic parameters could be correlated with the higher growth rates observed in P. pagrus acclimated to BW, while no significant effects due to the stocking density used were observed. [ABSTRACT FROM AUTHOR]

We tested the possibility of measuring progesterone levels in bubbler samples collected from free-ranging live bottlenose dolphins ( n = 11) and long-finned pilot whales ( n = 2) as a tool to evaluate the pregnancy status of individuals. Samples were collected during January 2004 and September-October 2005 in the Strait of Gibraltar and Gulf of Cadiz and used for sex-determination by genetic methods (skin samples) and for quantification of progesterone levels by enzyme immunoassay. Photo-identification tracking of females after taking the biopsy was used to observe the presence of newborns and, in this way, to determine if the female was pregnant at the time of sampling. Mean progesterone levels from pregnant bottlenose dolphins ( n = 2) were around 9 times higher than those from non-pregnant females ( n = 9), with no overlap between concentration ranges demonstrating that this method could constitute an effective tool for determining pregnancy in wild populations of bottlenose dolphins and other cetacean species. [ABSTRACT FROM AUTHOR]

Abstract: Mexel®432 is an anionic surfactant used as biocide in the cooling water system of power plants for biofouling reduction. Refrigeration waters from power plants do not usually follow, prior to their discharge to sea, any treatment aimed to eliminate biocides and this can have negative consequences on the aquatic fauna nearby. The effects of different concentrations of the antifoulant Mexel®432 (0.5, 1 and 2mgL−1) on osmoregulation (osmolality and Na+/K+-ATPase activity) and stress parameters (cortisol, glucose, and lactate) were assessed in juveniles of the flatfish Solea senegalensis. Gill histopathology and alterations due to oxidative stress (increased lipid peroxidation, LPO, levels) at branchial and hepatic levels were also considered. Other parameters tested were the antioxidant enzymes (catalase, CAT; glutathione peroxidase, GPX; and glutathione reductase, GR), xenobiotic metabolism defenses involved in detoxification (carboxylesterase, CbE; 7-ethoxyresorufin O-deethylase, EROD; and glutathione S-transferase, GST) and the neurotransmitter acetylcholinesterase (AChE) activity. Significant variations in osmoregulatory parameters, histological lesions and decreased branchial Na+/K+-ATPase activity were observed in exposed fish. Other gill biomarkers presented little or no significant variations in relation to controls. In contrast, hepatic parameters, such as CAT activity, were inhibited while EROD activity was initially elevated but after longer exposures it recovered basal values. These results suggested that under our experimental protocol exists toxic associated to Mexel®432 exposures. [Copyright &y& Elsevier]

Abstract: The time–course stress responses (0, 1, 2, and 7 d) was assessed in plasmatic, branchial and renal parameters of juveniles Solea senegalensis exposed to different concentrations of the antifouling sodium hypochlorite (0.1, 0.2, and 0.5mgL−1). These stress responses were only assessed for the total length of exposure (7d) at the lowest NaClO concentration due to the high toxicity of this chemical. In addition, the xenobiotic metabolism responses were evaluated by means of enzymatic activities of ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPX), catalase (CAT), and carboxylesterase (CbE) in liver; as well as GST, GPX, CAT and acetylcholinesterase (AChE) in gill. Oxidative stress damage due to sodium hypochlorite exposure was measured by lipid peroxidation levels in liver and gill. Concentrations of 0.2 and 0.5mgL−1 produced lethal effects after 1d and 2h of exposure, respectively. After 1d of exposure to sublethal concentration of sodium hypochlorite (0.1mgL−1) osmoregulatory (osmolality and chloride) and stress (cortisol, glucose and lactate) plasmatic parameters were enhanced to respect at control fish. However after 3 or 7d these parameters returned to control values. No effects were observed on plasma protein and triglyceride levels or on gill and kidney Na+/K+-ATPase activities. Diverse gill pathologies such as hypertrophy, lamellar fusion and an increase in goblet cell number and size were observed after 7d of exposure. Most biochemical parameters related to xenobiotic metabolism and oxidative stress were also significantly affected which suggests that seawater affected by sodium hypochlorite discharges from power plants, is able to alter the fish xenobiotic metabolism and generate oxidative stress. [Copyright &y& Elsevier]

Previous studies have shown that Senegalese sole is partially euryhaline in the juvenile phase, being able to adapt to a wide range of salinities in a short-time period, due to changes at the osmoregulatory and metabolic level. This study aimed to assess the effects of acclimation of sole to a wide range of salinities, with a special emphasis on the role of plasma amino acids during this process. Sole juveniles were acclimated for 2 weeks to different salinities: 5, 15, 25, 38, and 55 g L−1. Plasma levels of cortisol, glucose, osmolality, and free amino acids were assessed at the end. Changes in plasma levels of cortisol, glucose, and amino acids indicate that fish reared at 5 and 55 g L−1 were facing extra energy costs. Amino acids seem to play an important role during salinity acclimation, either as energy sources or as important osmolytes for cell volume regulation. [ABSTRACT FROM AUTHOR]

Seasonal variations in osmoregulatory and metabolic parameters were assessed in juvenile gilthead sea bream ( Sparus auratus) cultured in earthen ponds under a natural photoperiod and temperature. Specimens were sampled, and the plasma, gill, kidney and liver were collected during winter 2005 and 2006 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Plasma osmoregulatory parameters showed higher values in summer, while metabolic parameters presented different patterns of variations. Gill Na+,K+-ATPase activity decreased significantly in winter, while gill metabolite levels showed different patterns of variations among seasons. The enzymatic activities tested did not present a clear pattern of variation [(glutamate dehydrogenase (EC 1.4.1.2) (GDH) and hexokinase (EC 2.7.1.11) (HK)] or significant differences along seasons [glucose 6-phosphate dehydrogenase (EC 1.1.1.49)]. Kidney Na+,K+-ATPase activity decreased during summer and autumn. Different patterns of variation were observed in kidney metabolite levels while all the enzymatic activities assessed [lactate dehydrogenase-oxidase (EC 1.1.1.27) (LDH-O), HK and GDH] presented the highest values during summer. In the liver, metabolite levels and enzymatic activities did not show significant variations or present clear patterns of variation along different seasons. These results indicated seasonal variations in the osmoregulatory and metabolic parameters of different organs (blood, gill, kidney and liver) in earthen pond-cultured gilthead sea bream ( S. auratus), which could be mainly attributed to seasonal changes in temperature. [ABSTRACT FROM AUTHOR]

Simple Summary: The greater amberjack, Seriola dumerili, is of particular interest for pisciculture diversification due to its flesh quality and worldwide market acceptance. Moreover, this species shows rapid growth at the juvenile stage and 75% survival under captivity conditions. Among growing conditions, salinity is a critical factor for optimal growth. This study specifically assessed and characterized the physiological status of osmoregulation in individuals acclimated to three experimental salinities (15, 22, and 36 psu) in land-based recirculating aquaculture systems for 92 days. The results indicated several physiological adaptations to the different environmental salinities, both at the osmoregulatory and metabolic levels. Overall, our results suggest a beneficial effect of lower salinities for the juvenile stage to improve growth performance and avoid seawater-parasite outbreaks prior to transfer to sea cages. Osmotic costs in teleosts are highly variable, reaching up to 50% of energy expenditure in some. In several species, environmental salinities close to the isosmotic point (~15 psu) minimize energy demand for osmoregulation while enhancing growth. The present study aimed to characterize the physiological status related to osmoregulation in early juveniles of the greater amberjack, Seriola dumerili, acclimated to three salinities (15, 22, and 36 psu). Our results indicate that plasma metabolic substrates were enhanced at the lower salinities, whereas hepatic carbohydrate and energetic lipid substrates decreased. Moreover, osmoregulatory parameters, such as osmolality, muscle water content, gill and intestine Na+-K+-ATPase activities, suggested a great osmoregulatory capacity in this species. Remarkably, electrophysiological parameters, such as short-circuit current (Isc) and transepithelial electric resistance (TER), were enhanced significantly at the posterior intestine. Concomitantly, Isc and TER anterior-to-posterior intestine differences were intensified with increasing environmental salinity. Furthermore, the expression of several adeno-hypophyseal genes was assessed. Expression of prl showed an inverse linear relationship with increasing environmental salinity, while gh mRNA enhanced significantly in the 22 psu-acclimated groups. Overall, these results could explain the better growth observed in S. dumerili juveniles kept at salinities close to isosmotic rather than in seawater. [ABSTRACT FROM AUTHOR]

Abstract: Sexually immature Sparus auratus were injected intraperitoneally with coconut oil either alone (control) or containing 17β-estradiol (E2, 10 μg/g body mass) or 4-nonyphenol (4-NP, 100 and 200 μg/g body mass) and sampled 10 days later. Gill and kidney Na+,K+-ATPase activities, plasma levels of E2 and cortisol, plasma osmolites (osmolality, sodium and chloride) and metabolites (glucose, lactate, proteins and triglycerides) were examined. Livers were used for measuring hepatosomatic index (HSI) and determinations of the activities of antioxidant defences catalase (CAT) and total glutatione peroxidase (t-GPX), the CYP1A-dependent, 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST). HSI and plasma levels of E2 were significantly increased in E2-treated fish. E2 treatment enhanced plasma osmolality, glucose, triglycerides and proteins, but had no effect on plasma cortisol, and gill and kidney Na+,K+-ATPase activities. Hepatic activities of EROD, GST and CAT were significantly decreased after E2 administration, whereas t-GPX remained unaffected. Treatment with 200 μg/g 4-NP caused a slight increase in plasma E2 relative to the control group. Plasma glucose and protein levels were not affected by 4-NP, while triglycerides were increased. Fish treated with the higher dose of 4-NP displayed a clear reduction in kidney Na+,K+-ATPase activity, together with increases in plasma osmolality, relative to the control group. High 4-NP also caused a significant decrease in EROD and an increase in GST activity. Our results confirm the regulation of the natural estrogen E2 and the weak xenoestrogen 4-NP on osmoregulation and biotransformation enzymes in a partially similar manner. The actions of xenoestrogens on critical physiological processes may have an ecological significance as it can reduce adaptability and capacity to metabolise xenobiotics under stressful conditions. [Copyright &y& Elsevier]

Abstract: Gilthead sea bream (Sparus aurata) is a euryhaline species with a capacity to cope with demands in a wide range of salinities and thus is a perfect model-fish to study osmoregulatory responses to salinity-adaptive processes and their hormonal control. Immature sea bream acclimated to different salinities, i.e. SW (38‰), LSW (5‰) and HSW (55‰), were kept at 18 °C under natural photoperiod. Arginine vasotocin (AVT) and isotocin (IT) in plasma and pituitary were determined by HPLC. Plasma melatonin (Mel) was assayed by RIA. Plasma osmolality, ion concentrations (Na+, K+, Ca2+, Cl−) and Na+,K+-ATPase activity in gill were measured. A steady increase in plasma AVT, along with increasing water salinity was observed. Pituitary IT concentration in HSW-acclimated fish was significantly higher than that in LSW group. AVT/IT secretory system of sea bream does appear to be involved in the mechanism of long-term acclimation to different salinities. The distinct roles and control mechanisms of both nonapeptides are suggested. Plasma Mel was significantly higher in LSW compared with both HSW and SW groups. Data indicate that the changes in Mel level are linked to osmoregulation. Further studies are required to elucidate a complex role of AVT, IT and Mel in sea bream osmoregulation. [Copyright &y& Elsevier]

Simple Summary: Farmed fish must cope with different stressors during aquaculture procedures, such as high densities, fasting, transport, or air exposure during handling. The severity and timing of these stressors can produce important imbalances in the overall status of the animals, triggering several endocrine and physiological players. In this study, gilthead sea bream (Sparus aurata) juveniles were assigned to four experimental conditions: (1) fed at a low stocking density (LSD-F, 4 kg·m−3); (2) fed at a high stocking density (HSD-F, 40 kg·m−3); (3) food-deprived at LSD (LSD-FD); and (4) food-deprived at HSD (HSD-FD). This served to evaluate, both at the plasma and central (brain) levels, the role of several hormonal (cortisol and catecholamines) and monoamine (dopaminergic and serotonergic neurotransmitters) functionalities. Our results evidenced chronic stress exposure (i.e., a high stocking density and food deprivation) fallouts in the enhancement of the parameters related to the stress response, where monoaminergic activities in different brain regions served to reorganize the physiological response depending on the challenge applied. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: A fungal toxin, aflatoxin B1 (AFB1), undermines growth and stress axes of gilthead seabream (Sparus aurata) with depletion of somatic carbohydrate and lipid reservoirs. The present study assessed the physiological consequences of high stocking density versus low stocking density in seabream juveniles, which had previously been fed with AFB1 supplementation. These stressors are likely to converge by inferring animal welfare and economic profitability in the food animal industry. Interestingly, AFB1 seems to cause physiological and molecular dysfunction in response to overcrowding densities. Our results might be relevant to elucidate a potential risk for fish farming that is often overlooked. Several studies in fish have shown that aflatoxin B1 (AFB1) causes a disparity of species-dependent physiological disorders without compromising survival. We studied the effect of dietary administration of AFB1 (2 mg AFB1 kg−1 diet) in gilthead seabream (Sparus aurata) juveniles in combination with a challenge by stocking density (4 vs. 40 g L−1). The experimental period duration was ten days, and the diet with AFB1 was administered to the fish for 85 days prior to the stocking density challenge. Our results indicated an alteration in the carbohydrate and lipid metabolites mobilization in the AFB1 fed group, which was intensified at high stocking density (HSD). The CT group at HSD increased plasma cortisol levels, as expected, whereas the AFB1-HSD group did not. The star mRNA expression, an enzyme involved in cortisol synthesis in the head kidney, presented a ninefold increase in the AFB1 group at low stocking density (LSD) compared to the CT-LSD group. Adenohypophyseal gh mRNA expression increased in the AFB1-HSD but not in the CT-HSD group. Overall, these results confirmed that chronic AFB1 dietary exposure alters the adequate endocrinological physiological cascade response in S. aurata, compromising the expected stress response to an additional stressor, such as overcrowding. [ABSTRACT FROM AUTHOR]