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The aim of this study was to evaluate the effect of starvation and refeeding on the growth and food intake of gilthead seabream ( Sparus aurata ) and seabass ( Dicentrarchus labrax ) and on the growth and nitrogen uptake of glasswort ( Salicornia europaea ) in a polyculture aquaponic system under 12 ppt salinity for 75 days. Nine small-scale autonomous aquaponic systems were used, each containing 10 gilthead seabreams (average weight of 6.33 ± 0.73 g and average length of 5.73 ± 0.72 cm) and 10 seabasses (5.82 ± 0.77 g and 6.35 ± 0.45 cm), as well as five glasswort plants. Three fish feeding treatments were performed, a control (A), in which fish were fed daily until satiation, and two fasting treatments for 4 (B) and 7 days (C). Fish growth performance was significantly lower ( p  < 0.05) in the C treatment for both species compared to treatments A and B. Food consumption (FC) and feed conversion ratio (FCR) were significantly higher ( p  < 0.05) in treatment C. Glasswort growth performance was significantly higher in treatment C ( p  < 0.05). The results showed that the 4-day food-deprived fish were similar to the control fish by achieving partial compensatory growth. The more extended fasting period (7 days) resulted in significantly lower growth performance. The lipid and nitrogen retention levels in both species were significantly lower in food-deprived fish than in the control fish both before and during compensatory growth. The results suggest that a feeding schedule involving starvation-refeeding cycles is a promising feed management option for these species in polyculture aquaponic systems. The effect of food deprivation was also significantly beneficial ( p  < 0.05) for the growth performance of glasswort compared to the control treatment., Competing Interests: Efi Levizou is an Academic Editor for PeerJ., (©2024 Mitsopoulos et al.)

In the context of modern aquaculture, the effort to reduce the reliance on fishmeal/marine ingredients in fish diets has led to the exploration of plant-based protein sources as potential substitutes, a dietary shift that disrupts the bile acid profile in fish. Therefore, bile salts are being sought as additives. However, artificially increased intestinal levels of bile acids may significantly impact mucosal function. Therefore, here, we explored the regulatory role in the intestine of gilthead sea bream (Sparus aurata) of (i) chenodeoxycholic acid (CDC), (ii) a mixture formed by two bile acids, 3% cholic acid and 97% deoxycholic acid (MIX), and (iii) a conjugated bile salt sodium taurocholate (TC) in Ussing chambers with the epithelial voltage clamp technique. We tested the bile salts in a 50-500 μg/ml concentration range, and all of them promoted ion absorption. Yet, clear concentration-dependent and more pronounced effects on the ion transport were observed in the posterior intestine. On the other hand, bile salts had no or minor effects on tissue resistance. However, there are indications that the MIX could have adverse effects at high concentrations (500 μg/ml), promoting a threefold increase in tissue permeability measured using FITC-dextran (4 kD) regardless of the intestinal region, thus suggesting an alteration in intestinal permeability at high bile salt concentrations. The findings from our study emphasize the importance of considering intestinal function when contemplating the possible use of a particular bile salt as a dietary supplement. It appears that bile salts, whether acting individually or in combination, play a pivotal role in orchestrating nutrient absorption by influencing the function of epithelial ion transport. However further research is needed to fully grasp the region-dependent nuances of bile salt effects on ion transport and the ultimate consequences for nutrient absorption in the context of fish aquaculture., (© 2024. The Author(s).)

Classical assessments of new fish feeds are anthropocentric, focusing mainly on growth. Although this methodology is accurate, it does not consider the fish' perspective. This study aimed to investigate the behavioural responses and feed preferences of Nile tilapia (Oreochromis niloticus) and gilthead seabream (Sparus aurata) through a self-selection trial using self-feeders. Both species were offered three feeds: a control (PD) commercial-like feed and two diets (ORG1 and ORG2) formulated with different inclusions of alternative ingredients to address some of the current environmental concerns and/or ethical issues often associated with commercial formulations. Three groups of tilapia with an average weight of 163.0 g ± 4.3 g (mean ± SD) and four groups of seabreams with 174.7 g ± 27.0 g were tested. Tilapia exhibited a preference for ORG2 (46.5%), influenced by the sensory properties of the feed and post-ingestion signals. Seabream did not show a preference for any feed. These findings highlight the effectiveness of self-selection experiments in allowing fish to express their feeding behaviour and preferences. Therefore, this approach should be considered in the initial screening and design of new aquaculture feeds and ingredients., (© 2024. The Author(s).)

The contamination of marine and freshwater environments by nanoplastics is considered a global threat for aquatic biota. Taking into account the most recent concentration range estimates reported globally and recognizing a knowledge gap in polystyrene nanoplastics (PS-NPs) ecotoxicology, the present work investigated the harmful effects of 20 nm and 80 nm PS-NPs, at increasing biological complexity, on the rainbow trout Oncorhynchus mykiss RTG-2 and gilthead seabream Sparus aurata SAF-1 cell lines. Twenty nm PS-NPs exerted a greater cytotoxicity than 80 nm ones and SAF-1 were approximately 4-fold more vulnerable to PS-NPs than RTG-2. The engagement of PS-NPs with plasma membranes was accompanied by discernible uptake patterns and morphological alterations along with a nuclear translocation already within a 30-min exposure. Cells were structurally damaged only by the 20 nm PS-NPs in a time-dependent manner as indicated by distinctive features of the execution phase of the apoptotic cell death mechanism such as cell shrinkage, plasma membrane blebbing, translocation of phosphatidylserine to the outer leaflet of the cell membrane and DNA fragmentation. At last, functional analyses unveiled marked transcriptional impairment at both sublethal and lethal doses of 20 nm PS-NPs, with the latter impacting the "Steroid biosynthesis", "TGF-beta signaling pathway", "ECM-receptor interaction", "Focal adhesion", "Regulation of actin cytoskeleton" and "Protein processing in endoplasmic reticulum" pathways. Overall, a distinct ecotoxicological hazard of PS-NPs at environmentally relevant concentrations was thoroughly characterized on two piscine cell lines. The effects were demonstrated to depend on size, exposure time and model, emphasizing the need for a comparative evaluation of endpoints between freshwater and marine ecosystems., Competing Interests: Declaration of competing interest The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Low-impact fishing gear, such as fish pots, could help reduce human's impact on coastal marine ecosystems in fisheries but catch rates remain low and the harvest of resources used for baiting increases their environmental cost. Using black seabreams (Spondyliosoma cantharus) as target species in the Bay of Biscay, we developed and assessed the efficiency of biodegradable biopolymer-based baits (hereafter bio-baits) made of cockles (Cerastoderma edule) and different biopolymer concentrations. Through a suite of deep and machine learning models, we automatized both the tracking and behavior classification of seabreams based on quantitative metrics describing fish motion. The models were used to predict the interest behavior of seabream towards the bait over 127 h of video. All behavior predictions categorized as interested to the bait were validated, highlighting that bio-baits have a much weaker attractive power than natural bait yet with higher activity after 4 h once natural baits have been consumed. We also show that even with imperfect tracking models, fine behavioral information can be robustly extracted from video footage through classical machine learning methods, dramatically lifting the constraints related to monitoring fish behavior. This work therefore offers new perspectives both for the improvement of bio-baits and automatic fish behavior recognition., (© 2024. The Author(s).)

In recent decades, cadmium has emerged as an environmental stressor in aquatic ecosystems due to its persistence and toxicity. It can enter water bodies from various natural and anthropogenic sources and, once introduced into aquatic systems, can accumulate in sediments and biota, leading to bioaccumulation and biomagnification in the food chain. For this reason, the effects of cadmium on aquatic life remain an area of ongoing research and concern. In this paper, a multidisciplinary approach was used to assess the effects of long-term exposure to an environmental concentration on the hepatopancreas of farmed juveniles of sea bream, Sparus aurata. After determining metal uptake, metallothionein production was assessed to gain insight into the organism's defence response. The effects were also assessed by histological and ultrastructural analyses. The results indicate that cadmium accumulates in the hepatopancreas at significant concentrations, inducing structural and functional damage. Despite the parallel increase in metallothioneins, fibrosis, alterations in carbohydrate distribution and endocrine disruption were also observed. These effects would decrease animal fitness although it did not translate into high mortality or reduced growth. This could depend on the fact that the animals were farmed, protected from the pressure deriving from having to search for food or escape from predators. Not to be underestimated is the return to humans, as this species is edible. Understanding the behaviour of cadmium in aquatic systems, its effects at different trophic levels and the potential risks to human health from the consumption of contaminated seafood would therefore be essential for informed environmental management and policy decisions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Yeast, Saccharomyces cerevisiae, has been utilized as a probiotic in aqua-feeds to promote growth and alleviate the stress in aquatic animals. On the other hand, cadmium (Cd) toxicity causes serious retardation of growth and welfare status of aquatic animals. The present study was conducted to evaluate the protective role of dietary yeast in mitigating the waterborne Cd toxicity effects on the growth, haemato-biochemical, stress biomarkers, and histopathological investigations of gilthead seabream (Sparus aurata L.). In a 3 × 3 factorial design, the acclimated fish (20-24 g) were randomly distributed into nine treatments in triplicates where they were fed on 0.0% (control), 0.5%, and 1.0% of yeast along with exposure to 0.0, 1.0, and 2.0 mg Cd/L for 60 days. All growth parameters and mRNA expressions of IGF-1 and GH genes as well as haematological parameters were markedly increased with the increase of dietary yeast levels; meanwhile these variables were significantly retarded with Cd exposure. Contradictory effects on the above-mentioned variables were observed with Cd toxicity. In contrast, blood cortisol, glucose, total cholesterol, and triglyceride, lactate dehydrogenase, alanine transaminase, aspartate transaminase, alkaline phosphatase, in addition to DNA fragments % were noticeably increased with Cd toxicity especially at the treatment of 2.0 mg Cd/L, while decreasing with increasing dietary yeast levels. Compared with the control fish group, Cd concentrations in the gill, liver, and muscle tissues of gilthead seabream were higher in Cd-exposed treatments, especially at the treatment of 2.0 mg Cd/L. Deposition of Cd in fish liver was higher than that in gill tissues but lowest Cd residue was observed in muscle tissues. No significant changes in Cd residues in fish organs were observed in yeast-fed fish with no Cd exposure. The Cd exposure negatively affected histological status of gill, liver, and kidney tissues of S. aurata; while feeding Cd-exposed fish on yeast diets lowered the Cd residues in fish organs and recovered the adverse effects of Cd toxicity. Hence, this study recommends the addition of bakery yeast (1.0%) to fish diets to improve the performance, overall welfare, and histopathological status of gilthead seabream, S. aurata., (© 2023. The Author(s).)

This work investigated the effects of inorganic mercury (iHg) and methylmercury (MeHg) on the fish optic tectum morphology, viz. in relation to: (i) vulnerability of specific optic tectum layers; (ii) preferential targeting of Hg forms to neurons or glial cells; (iii) comparative toxicity of iHg and MeHg in this brain area that is in the maintenance of several fish behaviors. Two experiments exposing juvenile white seabream (Diplodus sargus) to waterborne iHg [HgCl 2 (2 μg L -1 )] and dietary MeHg (8.7 μg g -1 ) were performed, comprising both exposure (7 and 14 days; E7 and E14, respectively) and post-exposure (28 days; PE28) periods. Morphometric assessments were performed using stereological methods where the layers of the optic tectum were outlined, while its area and the number of neurons and glial cells were estimated. A histopathological assessment was also performed per section and per layer of optic tectum. iHg exposure did not trigger the loss of neurons during the exposure periods, while a decrease of glial cells was detected in a single layer of the optic tectum at E14. Differently, upon MeHg exposure, a decrease on the number of neurons and glial cells was found in several layers of optic tectum. In the post-exposure, both Hg forms triggered the loss of neurons, while only MeHg exposure led to a decrease on the number of glia cells. The histopathological assessment pointed out a higher toxicity of MeHg in the optic tectum layers, particularly in the post-exposure period, while no significant alterations were found in fish exposed to iHg. Hg forms targeted preferentially neurons. iHg and MeHg are relevant neurotoxicants to fish, with MeHg exposure leading to a higher toxicity than iHg in the optic tectum. After 28 days of post-exposure, iHg and MeHg neurotoxicity remained prominent, suggesting long-term effects of these toxicants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The role of subcutaneous adipose tissue adipocytes and the effects of fatty acids on carrageenan-induced skin inflammation in gilthead seabream (Sparus aurata) were studied. Fish were injected intramuscularly with phosphate-buffered saline (control) or λ-carrageenin (1%), and skin samples collected at the injection site at 3 and 6 h post-injection (p.i.) were processed for histological study. In addition, the presence and levels of lipid classes, fatty acid methyl esters (FAME) and eicosanoids were evaluated in the skin samples obtained from the injected areas. Histological results indicated an increase in adipocyte area in fish sampled at 3 h p.i. with λ-carrageenin compared to fish in the control group. Furthermore, the frequency of adipocytes between 4500 and 5000 μm 2 was increased at 6 h in the λ-carrageenin group compared to the control group. Analysis of lipid classes found that fish injected with λ-carrageenan showed increased free fatty acid (FFA) and sphingomyelin content at 3 and 6 h, respectively, compared to the control group. An increase in saturated fatty acids (SFA), n-6 polyunsaturated fatty acids (PUFA), and a decrease in the values of monounsaturated fatty acids (MUFA), n-3 PUFA and minor fatty acids were observed in fish skin at 6 h after λ-carrageenin injection, with respect to the values obtained in the control group. Regarding the analysis of eicosanoids, an increase in hydroxyeicosatetraenoic acid (5-HETE) was detected in the skin of fish at 6 h post-carrageenin injection compared to the control group. The presented results indicate the contribution of adipocytes and fatty acids in the development and regulation of the inflammatory response triggered by λ-carrageenin in gilthead seabream skin., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In oviparous marine fishes, the hyperosmotic induction of sperm motility in seawater (SW) is well established, however, the potential function of ion channels in the maintenance of post activated spermatozoon swimming performance remains largely unknown. Here, we investigated the influence of ion channels on the spermatozoon swimming parameters using the gilthead seabream ( Sparus aurata ) as a model for modern marine teleosts. Our data show that the SW-induced activation of seabream sperm motility requires three concomitant processes, the hyperosmotic shock, an ion-flux independent increase of the intracellular concentration of Ca 2+ ([Ca 2+ ] i ), but not of [K + ] i or [Na + ] i , and the alkalization of the cytosol. The combination of all three processes is obligatory to trigger flagellar beating. However, the time-course monitoring of sperm motion kinetics and changes in the [Ca 2+ ] i , [K + ] i and [Na + ] i in SW or in non-ionic activation media, showed that the post activated maintenance of spermatozoa motility is dependent on extracellular Ca 2+ and K + . A meta-analysis of a seabream sperm transcriptome uncovered the expression of multiple ion channels, some of which were immunolocalized in the head and/or tail of the spermatozoon. Selective pharmacological inhibition of these ion channel families impaired the long-term motility, progressivity, and velocity of SW-activated spermatozoa. The data further revealed that some antagonists of K + -selective or Ca 2+ -selective channels, as well as of stretch-activated and mechanosensitive channels, altered the trajectory of spermatozoa, suggesting that these ion channels are likely involved in the control of the swimming pattern of the post activated spermatozoon. These combined findings provide new insight into the signaling pathways regulating spermatozoon activation and swimming performance in marine fishes.

Haematological parameters are frequently used as physiological indicators in aquaculture studies. These parameters also have extended applications in clinical evaluation, diagnosis and prognosis in fish health status. However, no normal reference range of values has been demonstrated in depth for any of these haematological parameters for the European sea bass (Dicentrarchus labrax) or gilthead seabream (Sparus aurata). The main objective of this article is to present for the first time through extended literature review, the haematological parameters normal range values for the two most important aquaculture fish species farmed in Mediterranean Sea, D. labrax and S. aurata, and to demonstrate their similarities and their differences. In this article, we also discuss the environmental and external factors affecting their normal blood parameters values and we propose fundamental guidelines on the reporting units., (© 2022 John Wiley & Sons Ltd.)

Very long-chain (> C24) polyunsaturated fatty acids (VLC-PUFA) play an important role in the development of nervous system, retinal function and reproductive processes in vertebrates. Their presence in very small amounts in specific lipid classes, the lack of reference standards and their late elution in chromatographic analyses render their identification and, most important, their quantification, still a challenge. Consequently, a sensitive and feasible analytical methodology is needed. In this work, we have studied the effect of chain length, as well as the number and position of unsaturations (or double bonds) on the response of GC-APCI-(Q)TOF MS, to establish an analytical method for VLC-PUFA quantification. The developed methodology allows the quantification of these compounds down to 2.5 × 10 -3  pmol/mg lipid. The reduction of VLC-PUFA levels in lipid fractions of the organs from the herein sampled farmed fish suggesting a yet undetected effect on these compounds of high vegetable oil aquafeed formulations, that currently dominate the market., (© 2022. The Author(s).)

The current work aimed to carry out an in vivo study of the λ-carrageenin-induced inflammation in the skin of gilthead seabream (Sparus aurata). The fish were injected intramuscularly with phosphate-buffered saline (PBS, as control) or λ-carrageenin (1% in PBS), and the injection zone was evaluated by real-time ultrasonography (Vevo Lab, VisualSonics) at 1.5, 3, 6, 12, and 24 h post-injection (p.i.). Results demonstrated that the skin thickness was increased in fish injected with λ-carrageenin and sampled at 1.5, 3, and 6 h p.i. However, the skin thickness of the injected area decreased to the normal values in those fish sampled at 12 and 24 h p.i. In addition, fish injected with λ-carrageenin and analysed at 1.5, 3, and 6 h p.i. showed, in the underlying muscle at the injection place, several hyperechoic small foci surrounded by an anechoic area which were not observed in control fish. Furthermore, the fish were analysed by X-ray micro-computed tomography (micro-CT). The analysis of the micro-CT acquisitions revealed also a dark area in the place of the injection with λ-carrageenin at 1.5, 3, and 6 h. These areas were smaller in fish analysed at longer times (12 h p.i.) and were almost disappeared in fish sampled at 24 h p.i. These areas had an average density of -850 to -115 HU, which did not correspond with any tissue density of the rest of the body. Furthermore, similar dark areas at the injection zones were never observed in control fish. Present results support the use of both non-invasive techniques to study the inflammatory process in fish of commercial interest such as gilthead seabream., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nanoparticles (NP) are potentially reprotoxic, which may compromise the success of populations. However, the reprotoxicity of NP is still scarcely addressed in marine fish. Therefore, we evaluated the impacts of environmentally relevant and supra environmental concentrations of titanium dioxide (TiO 2 : 10 to 10,000 μg·L -1 ) and silver NP (Ag: 0.25 to 250 μg·L -1 ) on the sperm of gilthead seabream (Sparus aurata). We performed short-term direct exposures (ex vivo) and evaluated sperm motility, head morphometry, mitochondrial function, antioxidant responses and DNA integrity. No alteration in sperm motility (except for supra environmental Ag NP concentration), head morphometry, mitochondrial function, and DNA integrity occurred. However, depletion of all antioxidants occurred after exposure to TiO 2 NP, whereas SOD decreased after exposure to Ag NP (lowest and intermediate concentration). Considering our results, the decrease in antioxidants did not indicate vulnerability towards oxidative stress. TiO 2 NP and Ag NP induced low spermiotoxicity, without proven relevant ecological impacts., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Endocrine-disrupting chemicals include natural and synthetic estrogens, such as 17α-ethynilestradiol (EE 2 ), which can affect reproduction, growth and immunity. Estrogen signalling is mediated by nuclear or membrane estrogen receptors, such as the new G-protein-coupled estrogen receptor 1 (GPER1). The present work studies the effect of EE 2 and G1 (an agonist of GPER1) on body and muscle parameters and growth-related genes of 54 two-year-old seabreams. The fish were fed a diet containing EE 2 (EE 2 group) and G1 (G1 group) for 45 days and then a diet without EE 2 or G1 for 122 days. An untreated control group was also studied. At 45 days, the shortest body length was observed in the G1 group, while 79 and 122 days after the cessation of treatments, the shortest body growth was observed in the EE 2 group. Hypertrophy of white fibers was higher in the EE 2 and G1 groups than it was in the control group, whereas the opposite was the case with respect to hyperplasia. Textural hardness showed a negative correlation with the size of white fibers. At the end of the experiment, all fish analyzed in the EE 2 group showed a predominance of the gonadal ovarian area. In addition, the highest expression of the mafbx gene (upregulated in catabolic signals) and mstn2 (myogenesis negative regulator) was found in EE 2 -exposed fish.

The gut-brain axis plays a major role in multiple metabolic regulation processes, but studies regarding its responses to environmental stress in fish are still limited. In this study, we performed transcriptome sequencing analysis and enzyme-linked immunosorbent assay (ELISA) in yellowfin seabream (Acanthopagrus latus) exposed to environments with different water salinity (freshwater: 0 ppt; low-saline water: 3 ppt; brackish water: 6 ppt). According to transcriptome analysis, 707 and 1477 genes were identified as differentially expressed genes (DEGs) between freshwater and brackish water treatments in the brain and gut, respectively. Brain DEGs were significantly enriched into a set of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with signal transduction, most of which were downregulated. Gut DEGs were enriched into a neurotransmission-relevant KEGG pathway tryptophan metabolism, and the downregulated DEGs were enriched into the KEGG pathway focal adhesion. ELISA demonstrated significant physiological responses of the brain and gut across treatments, as determined by the concentrations of tight junction protein ZO-2, interleukin 1β, and serotonin. Under hypoosmotic stress, the functions of the gut-brain axis are altered via impairment of intestinal barrier integrity, by disturbance of gut-brain neurotransmission, and through tissue-damaging inflammatory reactions. Our work identified candidate genes which showed significantly differential expression in the gut-brain axis when yellowfin seabream encountered hypoosmotic stress, which could shed lights on the understanding of the potential osmotic regulation mechanisms of the gut-brain axis in teleost., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Alternative nutrient sources to fishmeal for fish feed, such as insect meals, represent a promising sustainable supply. However, the consequences for fish digestive function have not been exhaustively investigated. In the present study we evaluated the effect of partial fishmeal substitution with 10% Hermetia illucens (Hi10) larvae meal on the neuromuscular function of proximal and distal intestine in gilthead sea bream. In animals fed with insect meal, weight and growth parameters were similar to controls fed with conventional fishmeal. In addition, no anomalies in intestinal gross morphology and no overt signs of inflammation were observed. The gastrointestinal transit was significantly reduced in Hi10 fed animals. In the proximal and distal intestine longitudinal muscle, Hi10 feeding downregulated the excitatory cholinergic and serotoninergic transmission. Sodium nitroprusside-induced inhibitory relaxations increased in the proximal intestine and decreased in the distal intestine after Hi10 meal. Changes in the excitatory and inhibitory components of peristalsis were associated with adaptive changes in the chemical coding of both proximal and distal intestine myenteric plexus. However, these neuromuscular function alterations were not associated with considerable variations in morphometric growth parameters, suggesting that 10% Hi meal may represent a tolerable alternative protein source for gilthead sea bream diets., (© 2021. The Author(s).)

An important bottleneck in fish aquaculture research is the supply and maintenance of embryos, larvae, juvenile and adult specimens. In this context, cell lines represent alternative experimental models for in vitro studies that complement in vivo assays. This allows us to perform easier experimental design and sampling and avoid the sacrifice of animals. Embryonic stem (ES) cell lines have attracted increasing attention because they have the capability to proliferate indefinitely and could be differentiated into any cell type of the organism. To minimise cell heterogeneity and increase uniformity of in vitro studies results, in this manuscript we report the development and characterisation of two single cell-derived ES cell lines (monoclonal) from the morula stage embryos of the gilthead seabream, Sparus aurata, named as SAEC-A3 and SAEC-H7. Both cell lines have been passaged for over 100 times, indicating the establishment of long-term, immortalised ES cell cultures. Sequence analyses confirmed the seabream origin of the cell lines, and growth analyses evidenced their high viability and proliferating activity, particularly in culture medium supplemented with 10-15% fetal bovine serum and 22 °C. Both cell lines showed the ability to generate embryoid bodies and show different sensitivity and response to all-trans retinoic acid. The analysis of epithelial (col1α1) and neuronal (sox3) markers in differentiated cultures revealed that SAEC-A3 tended to differentiate towards epithelial-like cells whereas SAEC-H7 tended to differentiate towards neuronal-like cells. Both cell lines were efficiently transfected with pDsRed2-ER and/or pEGFP-N1 plasmids, indicating that they could represent useful biotechnological tools. Daily expression of pcna showed significant expression rhythms, with maximum levels of cell proliferation during the day-night transition. Currently, these cell lines are being successfully used as experimental models for the study of cellular metabolism, physiology and rhythms as well as for toxicological, pharmacological and gene expression analyses., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to find the relation of Sparus aurata (gilthead seabream) reproductive activities to some blood parameters as complete blood count, liver enzymes, some hormones related to reproduction process and microscopic findings of gonads, as well as expression of Bromodomain testis-specific gene. Eighty-eight sexually mature seabream were collected and investigated through the four seasons. Red blood cells were higher in autumn and spring. Hemoglobin was high in summer, MCV highest values ​​were seen in winter and summer, while MCHC was highest in summer. The values ​​of white blood cells increased significantly in spring, summer, and autumn compared with winter. The highest value of lymphocytes was recorded in spring and autumn. Eosinophil was recorded the highest value in the spring. The highest value of segmented neutrophils was recorded in summer. The highest value of band neutrophil was recorded in summer and winter. Alanine aminotransferase and aspartate aminotransferase showed high values in the winter. Luteinizing hormone (LH) was higher in females, males, and hermaphrodites during winter. Follicle-stimulating hormone (FSH) was higher in females during spring. The highest value of estradiol 17-β and progesterone was recorded in summer. The highest value of total testosterone was recorded in spring. Microscopically, ovaries were immature and inactive during spring and summer but well developed in autumn and winter. During spring and summer, testes were immature and began spermatogenesis process but well developed with the appearance of spermatids and spermatozoa during autumn and winter. The expression of Brdt was higher in testes than ovary. Brdt recorded high expression in autumn and spring than in summer and winter., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The effect of fasting on spawning performance, maternal, and egg nutrient composition, and on embryo/larval development was monitored in gilthead seabream (Sparus aurata). Two broodstocks were fasted during two consecutive years, for a period of 43 and 54 days within the spawning season, in a preliminary (year 1, 5-year-old breeders) and the main study (year 2, 6-year-old breeders), respectively. Mean daily fecundity showed a declining trend during fasting in the main study only, while fertilization success was high in both years and it was not affected by fasting, as was hatching and 5-day larval survival. There was a loss of 23.5% of maternal body mass due to fasting, and a reduction in gonadosomatic and hepatosomatic indexes, as well as crude protein in maternal muscle and gonads, but not in the liver. After fasting, muscle Σω-6 PUFA and C18:3ω-3 were reduced while C20:4ω-6, 20:5ω-3/20:4ω-6, and C22:6ω-3/20:4ω-6 increased; in the liver, significant reductions were observed in C16:0, C18:3 ω-3, 20:5ω-3/C22:6ω-3 and increases in C18:0, C20:5ω-3, Σω-6 PUFA, and 20:5ω-3/20:4ω-6; in gonads, C15:0, ΣMUFA, 20:5ω-3/C22:6ω-3, 20:5ω-3/20:4ω-6 were increased, while C18:1ω-9 and C20:5ω-3 decreased. Contrary to maternal tissues, the energy density and proximate composition of the eggs did not change due to fasting. The study suggests that fasting of gilthead seabream breeders for 6-8 weeks during the spawning period does not affect spawning performance, egg proximate composition, or embryo and early larval development since maternal nutrient reserves are mobilized to maintain optimal egg nutrient composition., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

This work aimed to carry out an in vivo study of the skin healing process in gilthead seabream (Sparus aurata) after being experimentally wounded. Firstly, the structure of normal skin was studied by real-time ultrasonography (Vevo Lab, VisualSonics) and light microscopy. Besides this, experimental wounds were made on the left flank of each fish with a circular biopsy punch (8 mm diameter) below the lateral line. The healing process was assessed on live fish at 0, 6, 11 and 23 days post-wounding using the real-time ultrasonography in B-mode and Power Doppler mode (Vevo 3100 FUJIFILM, VisualSonics). Through the ultrasonography images, both the skin structure and the evolution of the changes that wounds originated in the surrounding tissues were studied in vivo over time. Concomitantly, the pattern of neovascularization in the wounded area was followed during the healing process and it was demonstrated that, although the neovascularization started very early after the skin damage, it was increased in wounded areas from day 11 post-wounding onwards. The results obtained proved the utility and power of using ultrasounds in fish to evaluate in vivo complex biological processes in real time, which are difficult to study by other methodologies. The present data shed some light on the reparation of external injuries in aquatic vertebrates., (© 2021 John Wiley & Sons Ltd.)

Argyrops spinifer and Rhabdosargus haffara are two sympatric seabream species making important contributions to fisheries landings in the western Arabian/Persian Gulf. We identified the strengths and weaknesses in the long-term sustainability of A. spinifer and R. haffara stocks by integrating multiple sources of data, including fisheries catch and effort statistics, life history traits, scientific trawl surveys and historical length frequency distribution. Four strengths were identified in A. spinifer: wide distribution of juveniles, positive association to the network of de facto fishing exclusion areas created by hundreds of oil-gas facilities, early maturation and the existence of large and old individuals. A. spinifer suffers from two potential weaknesses: slow growth rate and higher exploitation pressure on the small-sized individuals. R. haffara, on the other hand, has a strength of having a short life span and a fast growth rate, characteristics that make it robust to unfavourable conditions. R. haffara suffers from two weaknesses: the lack of association to the oil and gas facilities, and the preference for nearshore shallow waters with stronger negative anthropogenic impacts. Identified strengths and weaknesses of these two sparids provided a preliminary assessment about their long-term sustainability, as well as a roadmap about how to develop different management strategies to meet specific objectives., (© 2021 Fisheries Society of the British Isles.)

Temperatures experienced during early ontogeny significantly influence fish phenotypes, with clear consequences for the wild and reared stocks. We examined the effect of temperature (17, 20, or 23 °C) during the short embryonic and yolk-sac larval period, on the swimming performance and skeleton of metamorphosing Gilthead seabream larvae. In the following ontogenetic period, all fish were subjected to common temperature (20 °C). The critical swimming speed of metamorphosing larvae was significantly decreased from 9.7 ± 0.6 TL/s (total length per second) at 17 °C developmental temperature (DT) to 8.7 ± 0.6 and 8.8 ± 0.7 TL/s at 20 and 23 °C DT respectively (p < 0.05). Swimming performance was significantly correlated with fish body shape (p < 0.05). Compared with the rest groups, fish of 17 °C DT presented a slender body shape, longer caudal peduncle, terminal mouth and ventrally transposed pectoral fins. Moreover, DT significantly affected the relative depth of heart ventricle (VD/TL , p < 0.05), which was comparatively increased at 17 °C DT. Finally, the incidence of caudal-fin abnormalities significantly decreased (p < 0.05) with the increase of DT. To our knowledge, this is the first evidence for the significant effect of DT during the short embryonic and yolk-sac larval period on the swimming performance of the later stages.

Oxygen uptake, heart rate and contraction frequencies of slow oxidative (SO) and fast glycolytic (FG) muscle were measured simultaneously in gilthead seabream Sparus aurata submitted to stepwise increases in current speed in a swimming respirometer. Variation in oxygen uptake was closely related to variation in heart rate, over initial steps these rose in concert with an increase in contraction frequency of SO muscle. There was an asymptote in oxygen uptake and heart rate at high speeds that reflected a transition from exclusive use of aerobic SO muscle to a combination of SO and anaerobic FG muscle, and which preceded fatigue., (© 2020 The Fisheries Society of the British Isles.)

Increasing global research has identified microplastics (MPs) to be impacting marine organisms. The present work aimed at investigating the physiological and behavioral effects of thirty-six juvenile Sparus aurata exposed to control, virgin and weathered MPs enriched diets during a 21-day period under controlled conditions. Physiological effects were assessed in liver and brain using the following biomarkers: activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRd), the detoxifying enzyme glutathione S-transferase (GST) and malondialdehyde (MDA) as indicative of lipid peroxidation. Individuals were recorded for behavior analysis (i.e. social interactions and feeding behavior). Results revealed an increase in cellular stress from control to weathered fish groups, with the virgin group showing intermediate levels in all quantified biomarkers. Significant differences were found in the liver for all biomarkers except for MDA, suggesting that exposure time to MPs in this experiment is long enough to trigger the activation of antioxidant enzymes but not to produce cell damage by lipid peroxidation. In brain tissue samples, fish from the weathered group presented significantly higher values for CAT and SOD, highlighting its function as primary antioxidants. Regarding behavioral effects, results showed that the two MPs enriched groups were significantly bolder during social interactions and, although no significantly, tended to be more active during feeding. In conclusion, MPs which have been weathered in marine environmental conditions produces a higher physiological response than virgin MPs but also, a physiological response is variable depending on the tissue analyzed. In addition, a short period to MP exposure seems to affect overall social and feeding behavior but, further research is needed to assess long-term effects of MP ingestion and its potential consequences on fish populations., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A major challenge in fish physiology is to understand the mechanisms underlying the transcriptomic responses of fish brain to food deprivation. Differential gene expression analysis identified in total 2240 transcripts that presented >2-fold change (adjusted p < 0.01) between each treatment and the control group (C). The identity of the transcripts was obtained with annotation against multiple public databases and they were grouped according to their expression patterns. The gene ontology terms that were substantially affected were identified by functional annotation analysis. Genes related to ion transport, cell cycle and cell adhesion were mainly regulated during fasting and refeeding. These findings contribute to identify key indicators for the molecular basis of brain functions during periods of starvation in gilthead seabream., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Living organisms have adapted to environmental oscillations in light and temperature through evolving biological clocks. Biological rhythms are pervasive at all levels of the endocrine system, including the somatotropic (growth) axis. The objective of the present research was to study the existence of daily rhythms on the somatotropic axis of a marine teleost species, specifically, the gilthead sea bream (Sparus aurata). Larvae of S. aurata at 30 dph (days post hatching), kept under a 9 L:15D (light-dark) photoperiod, were collected every 3 h throughout a 36 h cycle. The expression of the following somatotropic axis genes was analyzed by quantitative PCR: pituitary adenylate cyclase-activating polypeptide 1 (adcyap1), prepro-somatostatin-1 (pss1), growth hormone (gh), growth hormone receptor types 1 and 2 (ghr1 and ghr2, respectively), insulin-like growth factor 1 (igf1) and igf1 receptor a (igf1ra). All genes displayed significant differences among time points and, with the exception of adcyap1, all showed statistically significant daily rhythms. The acrophases of gh, ghr1, ghr2, igf1 and igf1ra were located around the end of the dark phase, between ZT19:44 and ZT0:48 h, whereas the highest expression levels of adcyap1 occurred at ZT18 h. On the other hand, the acrophase of pss1, an inhibitor of Gh secretion, was located at ZT10:16 h, hence it was shifted by several hours with respect to the other genes. The present results provide the first thorough description of somatotropic axis rhythms in gilthead sea bream. Such knowledge provides insights into the role of rhythmic regulation of the Gh/Igf1 axis system in larval growth and metabolism, and it can also improve the implementation of more species-specific feeding regimes., (Copyright © 2020. Published by Elsevier Inc.)

Leptin, ghrelin, and insulin influence lipid metabolism and thus can directly affect adipose tissue characteristics, modulating the organoleptic quality of aquaculture fish. The present study explored gilthead seabream (Sparus aurata) cultured preadipocytes development, and the regulation of adipogenesis by those three hormones. Preadipocytes presented a fibroblast-like phenotype during the proliferation phase that changed to round-shaped with an enlarged cytoplasm filled with lipid droplets after complete differentiation, confirming the characteristics of mature adipocytes. peroxisome proliferator-activated receptor-γ (pparγ) expression was higher at the beginning of the culture, while fatty acid synthase and 3-hydroxyacyl-CoA dehydrogenase gradually increased with cell maturation. The expression of lipoprotein lipase-like, lysosomal acid lipase (lipa), fatty acid translocase/cluster of differentiation-36 (cd36), and leptin receptor (lepr) were not affected during cell culture development; and undetectable expression levels were observed for leptin. Concerning regulation, leptin inhibited lipid accumulation significantly reducing pparγ and cd36 gene expression, both in early differentiating and mature adipocytes, while ghrelin decreased the expression of pparγ in the early differentiating phase but did not reduce intracellular lipid content significantly. Additional insulin past the onset of adipogenesis did not affect lipid accumulation either. In conclusion, at present culture conditions leptin has an anti-adipogenic function in differentiating preadipocytes of gilthead seabream and continues exerting this role in mature adipocytes, while ghrelin and insulin do not seem to influence adipogenesis progression. A better understanding of leptin, ghrelin, and insulin impact on the adipogenic process could help in the prevention of fat accumulation, improving aquaculture fish production and quality., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The interaction between diet and intestinal health has been widely discussed, although in vivo approaches have reported limitations. The intestine explant culture system developed provides an advantage since it reduces the number of experimental fish and increases the time of incubation compared to similar methods, becoming a valuable tool in the study of the interactions between pathogenic bacteria, rearing conditions, or dietary components and fish gut immune response. The objective of this study was to determine the influence of the total substitution of fish meal by plants on the immune intestinal status of seabream using an ex vivo bacterial challenge. For this aim, two growth stages of fish were assayed (12 g): phase I (90 days), up to 68 g, and phase II (305 days), up to 250 g. Additionally, in phase II, the effects of long term and short term exposure (15 days) to a plant protein (PP) diet were determined. PP diet altered the mucosal immune homeostasis, the younger fish being more sensitive, and the intestine from fish fed short-term plant diets showed a higher immune response than with long-term feeding. Vibrio alginolyticus ( V. alginolyticus ) triggered the highest immune and inflammatory response, while COX-2 expression was significantly induced by Photobacterium damselae subsp. Piscicida ( P. damselae subsp. Piscicida ), showing a positive high correlation between the pro-inflammatory genes encoding interleukin 1 β ( IL1-β ), interleukin 6 ( IL-6 ) and cyclooxygenase 2( COX-2 ).

Temperature variation affects the growth, maturation and distribution of fish species due to increasing constraints on physiological functions therefore, the aim of the present study is to evaluate effect of temperature on thermal tolerance and standard metabolic rate (SMR) of gilthead seabream (Sparus aurata). For this purpose, tolerable temperature ranges of juvenile gilthead seabream acclimated at 15, 20, 25, and 30 °C for 30 days were estimated using dynamic and static thermal methodologies. The SMRs of the fish were also determined based on oxygen consumption rate (OCR). The dynamic and static thermal tolerance zones of gilthead seabream were calculated as 737 °C 2 and 500 °C 2 , respectively, with a resistance zone area of 155.5 °C 2 . The SMR of the fish at the above acclimation temperatures (AT) was determined as 138, 257, 510, and 797 mg O 2 h -1 kg -1 , respectively and were significantly different (P < 0.01, n = 10). The temperature quotient (Q 10 ) in relation to the SMR of the fish was calculated as 3.45, 3.91, and 2.44 for acclimation temperature ranges of 15-20, 20-25, and 25-30 °C, respectively. The fact that the SMR increased with rising temperatures and then decreased gradually after 25 °C indicates that the temperature preference of juvenile gilthead seabream lies between 25 and 30 °C. This study shows that gilthead seabream tolerates a relatively narrow temperature range, and consequently, a low capacity for acclimatisation to survive in aquatic systems characterised by temperature variations., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The displacement of species from equatorial latitudes to temperate locations following the increase in sea surface temperatures is among the significant reported consequences of climate change. Shifts in the distributional ranges of species result in fish communities tropicalisation, i.e., high latitude colonisations by typically low latitude distribution species. These movements create new interactions between species and new trophic assemblages. The Senegal seabream, Diplodus bellottii, may be used as a model to understand the population genetics of these invasions. In the last decades, this species has undergone an outstanding range expansion from its African area of origin to the Atlantic coast of the Iberian Peninsula, where now occurs abundantly. Mitochondrial and nuclear markers revealed a striking high haplotypic nucleotide and genetic diversity values, along with significant population differentiation throughout the present-day geographical range of the Senegal seabream. These results are not consistent with the central-marginal hypothesis, nor with the expectations of a leptokurtic distribution of individuals, as D. bellottii seems to be able to retain exceptional levels of diversity in marginal and recently colonised areas. We discuss possible causes for hyperdiversity and lack of geographical structure and subsequent implications for fisheries.

Environmental enrichment is considered as a recommended tool to guarantee or improve the welfare of captive fish. This study demonstrates for the first time that structural environmental enrichment enhances cognition, exploratory behaviour and brain physiological functions of gilthead seabream (Sparus aurata). Seabream was reared in groups (n = 15) during 60 days under two different treatments: enriched tanks with plant-fibre ropes (EE) or bare/non-enriched tanks (NE). Fish were then exposed to a purpose-built maze for 1 h every second day in four trials. Analysis of video recordings showed that seabream under EE conditions presented higher overall exploratory behaviour, spatial orientation and learning capability compared to seabream from NE conditions. Results from brain monoamines analyses may suggest increased recent dopaminergic activity in telencephalon, known to be involved in learning processes; and increased serotonergic activity in cerebellum, involved in the coordination of balance, movements and orientation. In addition, EE-reared fish showed increased antioxidant activity in whole brain, with no apparent oxidative damage. Structural EE seemed to induce an hormetic response on juvenile seabream, improving their welfare status during captivity. Application of this kind of physical structure might be feasible at fish farms as a passive and non-invasive tool to improve welfare of intensively cultured seabream.

An 8-week feeding trial was conducted to evaluate the effect of replacement of fish meal (FM) with fish soluble meal (FSM) on growth performance, feed utilization and expression of genes involved in TOR signaling pathway for juvenile black sea bream (Acanthopagrus schlegelii). Six isonitrogenous (41%) and isolipidic diets were prepared to contain graded levels of FSM which replaced 0% (control diet), 10%, 20%, 30%, 40% and 60% protein from FM. Triplicate groups of 20 fish with initial weight 0.51 ± 0.01 g were fed with experimental diets twice daily to apparent satiation. The results showed significant differences in growth performance and feed utilization among all treatments, final body weight (FBW), percent weight gain (PWG), specific growth rate (SGR) and protein efficiency ratio (PER) significantly increased with dietary replacement levels of FM with FSM increasing from 0% to 40% (P < 0.05), PWG, SGR and PER were significantly reduced when replacement of FM with FSM further increased from 40% to 60%. Based on PWG against replacement levels of FM with FSM, A two-slope broken-line model analysis indicated that the optimal replacement of FM with FSM is to be 42.59%. Moreover, the lowest feed conversion ratio (FCR) was observed in fish fed the 40% FSM replacement diet. Muscle amino acid profile in muscle revealed that total essential amino acids, arginine and threonine were significantly influenced by replacement levels of FSM, while there was no significant difference in NEAA among all treatments. The hematological indices were not affected by the replacement levels of FM with FSM. The relative expression levels of irs-1, pi3k, akt, igf-1, s6k1 and tor were up-regulated when replacement levels of FM with FSM increased from 0% to 40%, and higher values were observed in fish fed with 40% FSM replacement diet compared to those fed the other diets. However, relative expression of 4e-bp2 was down-regulated when replacement levels of FM with FSM increased from 0% to 40% (P < 0.05). In summary, the results of present study indicated that FSM could be a viable alternative protein source for black sea bream, dietary FSM supplementation could improve growth and up-regulate the relative expression of irs-1, pi3k, akt, igf-1, s6k1 genes related to TOR signaling pathway in liver of juvenile black sea bream., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

17α-ethynilestradiol (EE 2 ) and tamoxifen (Tmx) are pollutants world-wide distributed in aquatic environments. Gilthead seabream, Sparus aurata L., is highlighted as a species model of intensively culture in anthropogenic disturbed environments. The effects of these pollutants on gilthead seabream reproduction and some immune responses have been described but, the humoral innate antimicrobial activities have never received attention. In this work we analysed the latest in the plasma of gilthead seabream males of different ages and reproductive stages treated with 0, 2.5, 5 or 50 μg EE 2 or 100 μg Tmx g -1 food during different times of exposure and of reverting to commercial diet (recovery). The peroxidase and protease activities decreased as the spermatogenesis of the first reproductive cycle (RC) proceeded in control fish. However, only protease and antiprotease activities showed different level at different stages of the second RC in control fish, but showed scarce disruption in fish treated with EE 2 or Tmx. Peroxidase and bactericide activities are more sensitive to EE 2 , than to Tmx. The effects induced by EE 2 varied depending on the activity analyzed, the dose and the time of exposure and the reproductive stage and the age of the specimens.

Background: This study evaluated the effects of partial substitution of dietary fishmeal (FM) with either fish protein hydrolysate (FPH) or autolysed dried yeast (HiCell®, Biorigin, Brazil) on intestinal microbiota of gilthead sea bream (Sparus aurata). A total number of 720 fish of 122.18 ± 6.22 g were fed for 92 days with three different diets in triplicate (3 tanks/diet). A diet based on FM/vegetable meal was used as control. The other two diets were formulated by replacing FM with 5% of either FPH or HiCell®. To analyze the gut microbiota associated to autochthonous and allochthonous microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME pipeline were used., Results: A total number of 102 OTUs (operational taxonomic units) at 97% identity were identified in fish gut samples collected at the end of feeding trial. Fourteen OTUs constituted the core gut microbiota, i.e. those OTUs found in at least nine out of fifteen samples per group and shared regardless of the diet. Eight OTUs were assigned to Firmicutes represented by Lactobacillus, Staphylococcus, and Bacillus genera, and six to Proteobacteria phylum. Dietary dried yeast autolysate modulated the intestinal microbiota by promoting the growth of some beneficial bacteria. At order level, fish fed yeast showed an enrichment in Bacillales and Clostridiales as compared to the control group, whereas fish fed FPH showed a significantly lower amount of bacteria belonging to Alteromonadales and Enterobacteriales than the other two feeding groups. Although we did not observe any effect of 5% FM replacement with alternative nitrogen sources at phylum level, at lower taxonomical levels, the composition of gut microbiota, in terms of relative abundance of specific taxa, was significantly influenced by the dietary treatment., Conclusions: The metabarcoding analysis revealed a clearly intestinal microbiota modulation in response to dietary autolyzed yeast. The abundance of some beneficial bacteria, i.e. indigestible carbohydrate degrading- and SCFA producing bacteria, was positively affected. Autolysed dried yeast obtained by the fermentation of a strain of Saccharomyces cerevisiae could be a valid alternative protein source to FM as well as a valid functional ingredient for aquafeed production [corrected].

Background: Aquaculture is a fast-growing industry and therefore welfare and environmental impact have become of utmost importance. Preventing stress associated to common aquaculture practices and optimizing the fish stress response by quantification of the stress level, are important steps towards the improvement of welfare standards. Stress is characterized by a cascade of physiological responses that, in-turn, induce further changes at the whole-animal level. These can either increase fitness or impair welfare. Nevertheless, monitorization of this dynamic process has, up until now, relied on indicators that are only a snapshot of the stress level experienced. Promising technological tools, such as proteomics, allow an unbiased approach for the discovery of potential biomarkers for stress monitoring. Within this scope, using Gilthead seabream (Sparus aurata) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used hormonal and metabolic indicators., Results: A broad spectrum of biological variation regarding cortisol and glucose levels was observed, the values of which rose higher in net-handled fish. In this sense, a potential pattern of stressor-specificity was clear, as the level of response varied markedly between a persistent (crowding) and a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net-handled fish had the highest number of differential proteins, compared to the other trials. Mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements of the response to stimulus., Conclusions: Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.

The effects of skin wounds on the intestinal barrier function and the beneficial effects of the dietary administration of Shewanella putrefaciens (known as SpPdp11) in gilthead seabream (Sparus aurata L.) were studied. Two replicates of fish were fed a commercial diet (control, CON) or CON diet enriched with 10 9  cfu g -1 SpPdp11 (SP diet) for 30 days. After this time, half of the fish were sampled, while the others were injured below the lateral line (wounded fish, W) and fed the same diets for an extra week before sampling (CON + W and SP + W groups). The intestinal histology and gene expression of different genes relevant for the intestinal barrier function were studied. The results showed that injured fish had a disordered enterocyte nucleus disposition, a more intense infiltration of mixed leucocytes and a thicker lamina propria in the intestine compared to the control fish. However, the fish in the SP + W group did not present these pathological symptoms in the intestine. No significant variations in the number of goblet cells were detected among the different experimental groups. Pro-inflammatory cytokines (colony-stimulating factor receptor 1, CSF1R, myeloperoxidase, MPO and interleukin-1β, IL-1β), mucins (intestinal mucin, IMUC and mucin 2, MUC2), and immunoglobulin T heavy chain (IGHT) were up-regulated, while tight junction protein occludin was down-regulated in the intestine from fish of the CON + W group. Similarly, the dietary administration of SpPdp11 markedly depressed the gene expression of pro-inflammatory cytokines, MUC2 and IGHT, but increased the gene expression of anti-inflammatory cytokine transforming growth factor-β1 (TGF-β1) and the tight junction proteins tricellulin and occluding after wounding. In brief, the skin wounds provoked an intestinal inflammatory response that included changes in the mucus layer and tight junction disruptions. Besides this, preventive administration of SpPdp11 alleviated the intestinal dysfunctions caused by skin wounds in gilthead seabream., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Origanum sp. is a very common genus of aromatic plants worldwide distributed around the Mediterranean area and O. vulgare (oregano) is the most important species of this genus throughout the world. Due the known medicinal properties of oregano, the effect of diets enriched with 0% (control), 0.5% and 1% oregano leaves powder was studied on the growth, immune and antioxidant status of gilthead seabream (Sparus aurata L.). Fish fed with oregano 0.5% and 1% enriched diets improved both humoral (IgM and bactericidal activity in skin mucus and protease activity in serum) and cellular (head kidney leucocytes phagocytic ability) immunity at 15 and 30 days. Furthermore, the addition of oregano did not provoke any significant effect neither in the growth promotion nor in the liver antioxidant enzymes activity studied in the serum and skin mucus. The possibility of using O. vulgare as a functional additive to fish diet is discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The gilthead seabream (Sparus aurata) is an important cultivated species in the Mediterranean area. A major problem for the gilthead seabream aquaculture sector derives from the high frequency of phenotypic abnormalities, including discolorations. In this study, we applied a whole-genome resequencing approach to identify a genomic region affecting a pigmentation defect that occurred in a cultivated S. aurata population. Two equimolar DNA pools were constructed using DNA extracted from 30 normally coloured and 21 non-pigmented fish collected among the offspring of the same broodstock nucleus. Whole-genome resequencing reads from the two DNA pools were aligned to the S. aurata draft genome and variant calling was performed. A whole-genome heterozygosity scan from single pool sequencing data highlighted a peak of reduced heterozygosity of approximately 5 Mbp on chromosome 6 in the non-pigmented pool that was not present in the normally coloured pool. The comparison of the non-pigmented with the normally coloured fish using a whole-genome F ST analysis detected three main regions within the coordinates previously detected with the heterozygosity analysis. The results support the presence of a major locus affecting this discoloration defect in this fish population. The results of this study have practical applications, including the possibility of eliminating this defect from the breeding stock, with direct economic advantages derived from the reduction of discarded fry. Other studies are needed to identify the candidate gene and the causative mutation, which could add information to understand the complex biology of fish pigmentation., (© 2020 Stichting International Foundation for Animal Genetics.)

The kisspeptin system, a known regulator of reproduction in fish, was investigated during two key phases within the gilthead seabream (Sparus aurata) life cycle: protandrous sex change and larval ontogeny. Seabream specific partial cDNA sequences were identified for two key targets, kissr4 and kiss2, which were subsequently cloned and qPCR assays developed. Thereafter, to examine association in expression with sex change, a group of adult seabream (2+ years old) undergoing sex change were sampled for gene expression at two different periods of the annual cycle. To study the kisspeptin system ontogeny during early life stages, transcript levels were monitored in larvae (till 30 days-post-hatch, DPH) and post-larvae (from 30 till 140 DPH). During sex change, higher expression of kissr4 and kiss2 was observed in males when compared to females or individual undergoing sex change, this is suggestive of differential actions of the kisspeptin system during protandrous sex change. Equally, variable expression of the kisspeptin system during early ontogenic development was observed. The higher expression of kissr4 and kiss2 observed from 5 DPH, with elevations at 5-20 and 90 DPH for kissr4 and at 5, 10, 20, and 60 DPH for kiss2, is coincident with the early ontogeny of gnrh genes previously reported for seabream, and possibly related with early development of the reproductive axis in this species., Competing Interests: Declaration of Competing Interest None. The funding providers had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Casting light on how the interaction between protection and density dependence affects fish population dynamics is critical for understanding the effectiveness of marine protected areas (MPAs). We developed a framework based on nonparametric statistics, model selection and multi-model inference to contrast alternative hypotheses about the effect of density dependence on demographic dynamics under protected and unprotected conditions. We trialed it using a 12-year long time series of white seabream (Diplodus sargus sargus) population density within the no-take zone of Torre Guaceto MPA (Italy) and at two nearby unprotected locations. Then, we showed how the demographic models obtained can be used to assess the consequences of protection on population viability. Population dynamics were significantly influenced by fish density within the MPA and at one of the unprotected locations, where demography is possibly driven by directional recruitment subsidy from the MPA. The comparison of population growth rates within and outside the MPA suggests that in unprotected conditions the fishery may remove a fraction between 40 and 70% of the population each year. The population viability analysis pointed out that, while the probability that the population becomes depleted (i.e. undergoes a local, temporary quasi-extinction) is high in unprotected locations, it is negligible within the no-take zone of the MPA., Competing Interests: The authors have declared that no competing interests exist.

In fish, as well as in other vertebrates, contrasting suites of physiological and behavioral traits, or coping styles, are often shown in response to stressors. However, the magnitude of the response (i.e. stress responsiveness) has been suggested to be independent of stress coping style. One central neurotransmitter that has been associated with both stress responsiveness and differences in stress coping styles is serotonin (5-hydroxytryptamine, 5-HT). In this study, we investigated to what extent stress responsiveness reflects differences in stress coping, and the potential involvement of the 5-HT system in mediating such differences in farmed Gilthead seabream. Initially, fish were classified as proactive or reactive based on their behavioural response to net restraint. Following 1.5 months, fish classified as proactive still showed a higher number of escape attempts and spent longer time escaping than those classified as reactive. These differences were reflected in a generally higher brain stem 5-HT concentration and a lower telencephalic 5-HT activity, i.e. the ratio of 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT, in proactive fish. Independent of stress coping styles, stress responsiveness was reflected in elevated 5-HIAA concentrations and 5-HIAA/5-HT ratios in telencephalon and brain stem together with increased plasma cortisol concentrations at 0.5 and 2 h following the last net restraint. The current results show that 5-HT signaling can reflect different behavioural output to a challenge which are independent of neuroendocrine responses to stress and lend support to the hypothesis that stress coping styles can be independent of stress responsiveness., (Copyright © 2019. Published by Elsevier Inc.)

Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psμ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na + ,K + -ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na + ,K + -ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (V max ) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na + ,K + -ATPase capacity appears fixed and activity appears to be determined by temperature., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The development of the marine aquaculture industry has led to the generation of significant amounts of fish wastes. Marine farm wastes exert adverse effects on the surrounding area of the cages. On the other hand, wastes of fish and other aquatic animals are regarded as major sources of valuable natural bioactive compounds, including enzymes, proteins, bioactive peptides, oil, amino acids, collagen, gelatin, calcium, biopolymers, and water-soluble minerals. To investigate the potential of marine fish waste, the whole digestive system of yellowfin seabream (Acanthopagrus latus) was extracted for extraction and identification of trypsin enzyme. Fish (179.93&plusmn;93.67 g; 184&plusmn;28.17 cm) were caught from the Persian Gulf and stored at -20 &deg;C. Yellowfin seabream were dissected and their whole digestive systems were removed. Samples were thoroughly washed with distilled water and purified through defatting using acetone and ammonium sulfate precipitation. The following issues were assessed: the total and specific activity of trypsin, protein determination, molecular weight, enzyme activity and stability in different pH values and temperatures. The obtained results indicated that specific activity and protein content of trypsin enzyme were 4.4 U and 3.4 mg/ml, respectively. The molecular weight of 23 kDa was reported for trypsin using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method. Maximum activity and stability of trypsin were observed at 60&deg;C and 45&deg;C, respectively. Trypsin demonstrated maximum activity and stability at a pH value of 8.0. In general, the results of the current study suggested that trypsin extracted from the digestive system of yellowfin seabream has considerable potential for industrial applications, such as the food industry, owing to its characteristics and stability under alkaline conditions., (Copyright © 2019, Archives of Razi Institute. Published by Kowsar.)

This study aimed to investigate the stress response of Sparus aurata specimens fed with nutraceutical aquafeed brewer's yeast (Saccharomyces cerevisiae) and spirulina (Arthrospira platensis). For that purpose, 96 (169.0 ± 2.8 g) animals were distributed randomly in 12 tanks (8 fish per tank, 4 replicates) and divided in 3 groups (D1, casein/gelatin, control; D2, brewer's yeast; D3, spirulina) and fed for 30 days. At the end of this period, fish from two replicates of each experimental diet were submitted to air exposure for 60 s while the fish from the other two replicates were maintained undisturbed (control). Afterwards, samples of blood, skin mucus, and head kidney were collected. The results revealed that after air exposure, cortisol, and glucose levels increased in the groups fed D1 (18.5 ± 2.6 mg/mL; 7.3 ± 0.6 mmol/L, respectively) and D2 (20.0 ± 6.2 mg/mL; 7.7 ± 0.6 mmol/L), but glucose not increased in fish fed D3 (13.7 ± 2.6 mg/mL; 5.5 ± 0.3 mmol/L). Lactate levels increased in all stressed groups, but in D1, its levels were significantly higher. After stress procedure, immunoglobulin M (IgM) levels in mucus increased only in fish fed D3 (0.1901 ± 0.0126 U/mL). Furthermore, there was a reduction in the expression of some genes involved in stress response (coxIV, prdx3, csfl-r, ucp1, and sod in fish fed D2 and D3). csf1 decreased only in stressed fish fed D2. However, cat increased in fish fed with D3. In summary, these findings points to the beneficial effects of spirulina and brewer's yeast to improve stress resistance in aquaculture practices of gilthead seabream.

The health of fishes from select aquacultures was investigated by conducting histopathologic and enzymatic analyses, as well as by examining pollutant accumulation rates in fish tissues ranging in age from juvenile to two years old. Histopathologic examinations demonstrated that the fishes had some abnormalities in their livers, spleens, intestines and reproduction systems, such as lipidation, ovotestis formation, lysis and enlargements of the tissues. The occurrence rate of these abnormalities was not very frequent but also not negligible. Statistical analysis demonstrated that enzyme activity (i.e. CAT, EROD, SOD) and protein concentration fluctuated predominantly by age and season. These parameters were not found to be related to the fish farm or other spatial changes, when their existing environmental conditions were not extreme (i.e. polluted or otherwise unsuitable). Metal concentrations (i.e. Ni, Cu, Zn, As, Cd, Pb and Hg) were never found to be higher than national or international regulatory limits. The quality of the fishes caught from optimal farm conditions may be evaluated as good quality for human consumption., (Copyright © 2019. Published by Elsevier Ltd.)

The survival of larval marine fishes during early development depends on their ability to feed before depleting their yolk reserves. Most larval fish capture prey by expanding their mouth, generating a 'suction flow' that draws the prey into it. These larvae dwell in a hydrodynamic environment that impedes their ability to capture even non-evasive prey; however, the marine environment is characterized by an abundance of evasive prey, predominantly copepods. Copepods sense the hydrodynamic disturbance created by approaching predators and perform high-acceleration escape maneuvers. Using a 3D high-speed video system, we characterized the interaction between Sparus aurata larvae and prey from a natural zooplankton assemblage that contained evasive prey, and assessed the factors that determine the outcome of these interactions. At 8-33 days post hatching, larvae preferentially attacked large prey that was moving prior to the initialization of the strike; however, feeding success was lower for larger, more evasive prey. Thus, larvae were challenged in capturing their preferred prey. Larval feeding success increased with increasing Reynolds numbers, but decreased sharply when the prey performed an escape maneuver. The kinematics of successful strikes resulted in a shorter response time but higher hydrodynamic signature available for the prey, suggesting that strike success in our experiments was determined by brevity rather than stealth: executing a fast strike eliminated a potential escape response by the prey. Our observations of prey selectivity reveal that larval performance, rather than preferences, determines their diet during early development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

In this study, we describe an abnormal ectopically mineralized structure (EMS) that was found inside the skull of a juvenile Sparus aurata that also showed a bilateral opercular deformation. The overall phenotype and tissue composition were studied using micro-CT scanning and histological analyses. The ectopic structure occupies a large volume of the brain cavity, partially extruding into the gill cavity. It shows a dense mineralization and an extracellular matrix-rich phenotype, with variation in both the morphology and size of the cell lacunae, combined with an irregular fibre organization inside the matrix. This study is the first to report such an EMS in a juvenile teleost fish, where the tissue does not resemble any other connective tissue type described in bony fish so far. The tissue phenotype seems to rule out that the EMS corresponds to a tumorous cartilage. Yet, it is rather reminiscent of a highly mineralized structure found in cartilaginous fish, where it is suggested to be associated with damage repair., (© 2019 John Wiley & Sons Ltd.)

We investigated links between swimming behavior and muscle bioenergetics in two emblematic Mediterranean fish species that have very different ecologies and activity levels. European sardines Sardina pilchardus are pelagic, they swim aerobically, school constantly and have high muscle fat content. Gilthead seabream Sparus aurata are bentho-pelagic, they show discontinuous spontaneous swimming patterns and store less fat in their muscle. Estimating the proportion of red and white muscle phenotypes, sardine exhibited a larger proportion of red muscle (~10% of the body mass) compared to gilthead seabream (~5% of the body mass). We firstly studied red and white muscle fiber bioenergetics, using high-resolution respirometers, showing a 4-fold higher oxidation capacity for red compared to white muscle. Secondly, we aimed to compare the red muscle ability to oxidize either lipids or carbohydrates. Sardine red muscle had a 3-fold higher oxidative capacity than gilthead seabream and a greater capacity to oxidize lipids. This study provides novel insights into physiological mechanisms underlying the different lifestyles of these highly-prized species., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Physiological rates and processes underpin the relationships between ectothermic organisms, such as fish, and their environment. The response and persistence of fish populations in an increasingly variable ocean is dependent on the distribution and diversity of physiological phenotypes. Growing evidence suggests that fisheries exploitation can selectively target certain physiological and behavioural phenotypes, which may shift exploited populations to altered physiological states. Here we test if commercial fisheries have the potential to do this in a "natural laboratory" along the South African coast. We compare metabolic traits of exploited and protected populations of the fish species, Chrysoblephus laticeps, which is a major component of the South African hook and line fishery. We find that high-performance aerobic scope phenotypes are reduced in the fished population. The most likely mechanism for this finding is a positive relationship between aerobic scope and capture vulnerability in passive-gear fisheries. Our results further highlight the selective nature of capture-fisheries and suggest that exploitation has the capacity to alter climate responses of fish populations on a physiological level. Our finding also implicates how Marine Protected Areas, through harbouring individuals with a greater diversity of physiological traits, may provide greater fish response diversity to environmental variability.