Dietary Debaryomyces hansenii promotes skin and skin mucus defensive capacities in a marine fish model.

The present study explores the effects of two supplementation levels of Debaryomyces hansenii (1.1% and 2.2%) as a probiotic in a reference low fish meal-based diet on the skin mucosal tissue in Sparus aurata. This study includes the evaluation of fish performance coupled with a holistic study of the skin mucosa: i) a transcriptomic study of the skin tissue, and ii) the evaluation of its secreted mucus both in terms of skin mucosal-associated biomarkers and its defensive capacity by means of co-culture analysis with two pathogenic bacteria. Results showed that after 70 days of diet administration, fish fed the diet supplemented with D. hansenii at 1.1% presented increased somatic growth and a better feed conversion ratio, compared to fish fed the control diet. In contrast, fish fed the diet including 2.2% of the probiotic presented intermediate values. Regarding gene regulation, the probiotic administration at 1.1% resulted in 712 differentially expressed genes (DEGs), among which 53.4% and 46.6% were up- and down-regulated, respectively. In particular, D. hansenii modulated some skin biological processes related to immunity and metabolism. Specifically, D. hansenii administration induced a strong modulation of some immune biological-related processes (61 DEGs), mainly involved in B- and T-cell regulatory pathways. Furthermore, dietary D. hansenii promoted the skin barrier function by the upregulation of anchoring junction genes (23 DEGs), which reinforces the physical defense against potential skin damage. In contrast, the skin showed modulated genes related to extracellular exosome and membrane organization (50 DEGs). This modulated functioning is of great interest, particularly in relation to the increased skin mucus defensive capacity observed in the bacterial co-culture in vitro trials, which could be related to the increased modulation and exudation of the innate immune components from the skin cells into the mucus. In summary, the modulation of innate immune parameters coupled with increased skin barrier function and cell trafficking potentiates the skin's physical barrier and mucus defensive capacity, while maintaining the skin mucosa's homeostatic immune and metabolic status. These findings confirmed the advantages of D. hansenii supplementation in low fish meal-based diets, demonstrating the probiotic benefits on cultured marine species.

Physiological Benefits of Dietary Lysophospholipid Supplementation in a Marine Fish Model: Deep Analyses of Modes of Action.

Given the hydrophilic structure of lysophospholipids (LPLs), their dietary inclusion translates into a better emulsifying capacity of the dietary components. The present study aimed to understand the mechanisms underlying the growth-promoting effect of LPL supplementation by undertaking deep analyses of the proximal intestine and liver interactomes. The Atlantic salmon (Salmo salar) was selected as the main aquaculture species model. The animals were divided into two groups: one was fed a control diet (C-diet) and the other a feed (LPL-diet) supplemented with an LPL-based digestive enhancer (0.1% AQUALYSO®, Adisseo). The LPL-diet had a positive effect on the fish by increasing the final weight by 5% and reducing total serum lipids, mainly due to a decrease in the plasma phospholipid (p < 0.05). In the intestine, the upregulated interactome suggests a more robust digestive capacity, improving vesicle-trafficking-related proteins, complex sugar hydrolysis, and lipid metabolism. In the liver, the LPL-diet promotes better nutrients, increasing several metabolic pathways. The downregulation of the responses to stress and stimuli could be related to a reduced proinflammatory state. This study on the benefits and modes of action of dietary LPLs opens a new window into fish nutrition and could be extended to other productive species.

Evaluating the Functional Properties of Spray-Dried Porcine Plasma in Gilthead Seabream (Sparus aurata) Fed Low Fish Meal Diets.

Blood by-products are an untapped source of high-quality ingredients for aquafeeds, containing a broad variety of cytokines, hormones, growth factors, proteins, bioactive peptides, and amino acids. The effects of the spray-dried porcine plasma (SDPP), a type of processed animal protein on several immune parameters, were evaluated in sea bream using ex vivo and in vitro assays. In this study, fish were fed with two isoproteic, isolipidic, and isoenergetic diets: control diet (7% fish meal, FM) and SDPP diet (2% FM and 5% SDPP). At the end of the 92-days trial, those fed the SDPP diet were larger in body weight (p < 0.05) without differences in feed conversion ratio (p > 0.05). The ex vivo immune stimulation of splenocytes indicated that SDPP had a beneficial effect in promoting systemic immunity, since the surface cell marker (cd4), pro- (il-1ß), and anti-inflammatory (tgf-ß1) cytokines, and genes involved in humoral immunity (IgM) were up-regulated. The co-culture assays of skin mucus corroborated that SDPP enhanced the antibacterial capacity of mucus against V. anguillarum. In addition, main mucus biomarkers did not show significant differences, except for cortisol levels which were lower in the SDPP diet. The present study indicated that SDPP may be considered a functional ingredient in aquafeeds formulated with low FM levels.

Effects of Acute Psychological and Physiological Stress on Rock Climbers.

BACKGROUND: The aim of this study was to assess the effects that psychological and physiological stressors have on indoor rock climbers, as well as to identify sex differences. METHODS: 14 intermediate rock climbers participated in the study, 10 males and 4 females. Mean age was 31 ± 8 years for males and 21 ± 2 years for females. Day 1 consisted of test familiarization and baseline measurements. Day 2 included two test conditions, startle and fatigue, separated by 20 min. In the startle condition, participants had to lead climb a route, and a loud audio stimulus was presented near the top of the climb. In the fatigue condition, participants were required to climb as fast as they could until muscular failure. The competitive state anxiety inventory second review (CSAI-2R) questionnaire was used to assess somatic anxiety, cognitive anxiety, and self-confidence. The four-square step test (FSST) was used to assess motor control, and cortisol levels were acquired via passive drool (PD). RESULTS: Cortisol concentrations were highest in the pre-startle condition (1.72 µg/dL ± 0.66), and values decreased post-startle (1.67 µg/dL ± 0.74) and post-fatigue (1.42 µg/dL ± 0.72). However, cortisol concentrations increased post-startle in females (1.57 µg/dL ± 0.96). Somatic anxiety in males was significantly higher post-startle (16.36 ± 5.54) than pre-startle (14.23 ± 5.09). Females had significantly higher somatic anxiety post-startle (18.00 ± 8.76), and they had lower self-confidence levels (30.00 ± 5.89) than males. CONCLUSIONS: There are differences in the way that males and females prepare and respond to stressful situations. Furthermore, time of day may have had a significant impact on cortisol concentrations.

Impact of dietary porcine blood by-products in meagre (Argyrosomus regius) physiology, evaluated by welfare biomarkers and the antibacterial properties of the skin mucus.

Tools are required for quick and easy preliminary evaluation of functional feeds efficiency on fisheries. The analysis of skin mucus biomarkers is a recent alternative approach providing a faster feed-back from the laboratory which is characterized by being less invasive, more rapid and with reduced costs. The effect of replacing fishmeal and fish protein hydrolysates by means of two porcine by-products, the porcine spray-dried plasma (SDPP) and pig protein hydrolysate (PPH), in compound diets (50.4% crude protein, 16.2% crude protein, 22.1 MJ/kg feed) was evaluated in juvenile meagre (Argyrosomus regius) during a two-months period. To determine the impact of these dietary replacements, growth and food performance were measured together with digestive enzymes activities and filet proximal composition. Additionally, skin mucus was collected and characterized by determining main mucus biomarkers (protein, glucose, lactate, cortisol, and antioxidant capacity) and its antibacterial properties, measured by the quick in vitro co-culture challenges. In comparison to the control group, the inclusion of PPH and SDPP, in meagre diets reduced growth (7.4-8.8% in body weight), increased feed conversion ratios (9.0-10.0%), results that were attributed to a reduction in feed intake values (24.2-33.0%) (P < 0.05). Porcine blood by-products did not modify the activity of gastric and pancreatic digestive enzymes as well as those involved in nutrient absorption (alkaline phosphatase) nor liver oxidative stress condition (P > 0.05). In contrast, a reduction in fillet lipid content associated to an increase in fillet protein levels were found in fish fed SDPP and PPH diets (P < 0.05). As compared to the control diet, the dietary replacement did not alter the levels of the skin mucus biomarkers related to stress (cortisol and antioxidant capacity) or nutritional status (soluble protein, glucose and lactate) (P > 0.05). Interestingly, regardless of the worst performance in somatic growth, meagre fed diets containing both tested porcine by-products showed a significantly improved antibacterial capacity of their skin mucus. This enhancement was more prominent for fish fed with the PPH diet, which may be attributed to a higher content of immunomodulatory bioactive compounds in PPH. Further research will be necessary to provide insights on how the inclusion of SDPP and PPH, at the expense of dietary fishmeal and fish protein hydrolysates, affects feed intake and growth performance in meagre. However, the use of skin mucus biomarkers has been demonstrated to be an excellent methodology for a preliminary characterization of the functional feeds, in particular for their prophylactic properties by the study of mucus antibacterial activity.

Porcine Protein Hydrolysates (PEPTEIVA®) Promote Growth and Enhance Systemic Immunity in Gilthead Sea Bream (Sparus aurata).

The effects of porcine plasma protein hydrolysate (PPH) on growth, feed efficiency, and immune responses was evaluated in Sparus aurata. Fish were fed two isoproteic (48% protein), isolipidic (17% fat), and isoenergetic diets (21.7 MJ/kg) diets, one of them containing 5% PPH at the expense of fishmeal. Both diets were tested for 92 days. A significant increase in growth was observed in fish fed the PPH diet in comparison to the control group (182.2 ± 4.4 vs. 173.8 ± 4.1 g), as well as an increase in feed intake without worsening FCR values. An ex vivo assay, with splenocytes incubated with lipopolysaccharide, was conducted to evaluate the cellular immune competence of fish. Genes involved in humoral immunity (lys, IgM), pro- (tnf-α, il-1ß), and anti-inflammatory (tgf-ß1, il10) cytokines were upregulated in the PPH group in comparison to the control group. The inclusion of PPH in diets enhanced the antibacterial capacity of skin mucus, as the co-culture of selected bacteria (E. coli, V. anguillarum, and P. anguilliseptica) with skin mucus indicated. The present results showed that the PPH in low fishmeal diets (2%) promoted growth and feed efficiency, as well as enhancing the immune response, which indicates that this is a safe and functional ingredient for aquafeeds.

Environmental Salinity Modifies Mucus Exudation and Energy Use in European Sea Bass Juveniles.

The European sea bass (Dicentrarchus labrax) is a euryhaline marine teleost that can often be found in brackish and freshwater or even in hypersaline environments. Here, we exposed sea bass juveniles to sustained salinity challenges for 15 days, simulating one hypoosmotic (3‰), one isosmotic (12‰) and one hyperosmotic (50‰) environment, in addition to control (35‰). We analyzed parameters of skin mucus exudation and mucus biomarkers, as a minimally invasive tool, and plasma biomarkers. Additionally, Na+/K+-ATPase activity was measured, as well as the gill mucous cell distribution, type and shape. The volume of exuded mucus increased significantly under all the salinity challenges, increasing by 130% at 50‰ condition. Significantly greater amounts of soluble protein (3.9 ± 0.6 mg at 50‰ vs. 1.1 ± 0.2 mg at 35‰, p < 0.05) and lactate (4.0 ± 1.0 µg at 50‰ vs. 1.2 ± 0.3 µg at 35‰, p < 0.05) were released, with clear energy expenditure. Gill ATPase activity was significantly higher at the extreme salinities, and the gill mucous cell distribution was rearranged, with more acid and neutral mucin mucous cells at 50‰. Skin mucus osmolality suggested an osmoregulatory function as an ion-trap layer in hypoosmotic conditions, retaining osmosis-related ions. Overall, when sea bass cope with different salinities, the hyperosmotic condition (50‰) demanded more energy than the extreme hypoosmotic condition.

Carvacrol, Thymol, and Garlic Essential Oil Promote Skin Innate Immunity in Gilthead Seabream (Sparus aurata) Through the Multifactorial Modulation of the Secretory Pathway and Enhancement of Mucus Protective Capacity.

One of the main targets for the use of phytogenics in aquafeeds is the mucosal tissues as they constitute a physical and biochemical shield against environmental and pathogenic threats, comprising elements from both the innate and acquired immunity. In the present study, the modulation of the skin transcriptional immune response, the bacterial growth capacity in skin mucus, and the overall health condition of gilthead seabream (Sparus aurata) juveniles fed a dietary supplementation of garlic essential oil, carvacrol, and thymol were assessed. The enrichment analysis of the skin transcriptional profile of fish fed the phytogenic-supplemented diet revealed the regulation of genes associated to cellular components involved in the secretory pathway, suggesting the stimulation, and recruitment of phagocytic cells. Genes recognized by their involvement in non-specific immune response were also identified in the analysis. The promotion of the secretion of non-specific immune molecules into the skin mucus was proposed to be involved in the in vitro decreased growth capacity of pathogenic bacteria in the mucus of fish fed the phytogenic-supplemented diet. Although the mucus antioxidant capacity was not affected by the phytogenics supplementation, the regulation of genes coding for oxidative stress enzymes suggested the reduction of the skin oxidative stress. Additionally, the decreased levels of cortisol in mucus indicated a reduction in the fish allostatic load due to the properties of the tested additive. Altogether, the dietary garlic, carvacrol, and thymol appear to promote the gilthead seabream skin innate immunity and the mucus protective capacity, decreasing its susceptibility to be colonized by pathogenic bacteria.

Multi-organ characterisation of B-esterases in the European sea bass (Dicentrarchus labrax): Effects of the insecticide fipronil at two temperatures.

In fish, the study of cholinesterases (ChEs) and carboxylesterases (CEs), apart from their involvement in neural activity and xenobiotic metabolism, respectively, requires to be further explored. The European sea bass (Dicentrarchus labrax) was the fish model used to characterise B-esterases in several matrices and organs, as well as to assess the impacts of the insecticide fipronil at two temperatures: the natural temperature at the time of sampling (13 °C) and at 16 °C (based on climate change-related predictions for the Mediterranean region). Fipronil exerts harmful effects in non-target species; however, some countries are reluctant to implement regulations without additional evidence on their toxicity. A comprehensive study was performed in fish pre-acclimated to the two targeted temperatures for 15 days. B-esterases were evaluated in multiple samples after 7 and 14 day exposures to fipronil in feed (dose of 10 mg/kg) and after a 7-day depurative period. Based on hydrolysis rates, results showed that CEs were measurable in all matrices while ChEs were more abundant in muscle and, particularly, acetylcholinesterase (AChE) in the brain. A + 3 °C increase in temperature had little influence on B-esterase activity; however, fipronil caused a significant increase in brain AChE (1.5-fold) and CE (3-fold) activities. Other matrices and organs also experienced alterations in their B-esterase activities that could compromise their physiological functions.

Evaluation of an Acute Osmotic Stress in European Sea Bass via Skin Mucus Biomarkers.

European sea bass is a marine teleost which can inhabit a broad range of environmental salinities. So far, no research has studied the physiological response of this fish to salinity challenges using modifications in skin mucus as a potential biological matrix. Here, we used a skin mucus sampling technique to evaluate the response of sea bass to several acute osmotic challenges (for 3 h) from seawater (35‱) to two hypoosmotic environments, diluted brackish water (3‱) and estuarine waters (12‱), and to one hyperosmotic condition (50‱). For this, we recorded the volume of mucus exuded and compared the main stress-related biomarkers and osmosis-related parameters in skin mucus and plasma. Sea bass exuded the greatest volume of skin mucus with the highest total contents of cortisol, glucose, and protein under hypersalinity. This indicates an exacerbated acute stress response with possible energy losses if the condition is sustained over time. Under hyposalinity, the response depended on the magnitude of the osmotic change: shifting to 3‱ was an extreme salinity change, which affected fish aerobic metabolism by acutely modifying lactate exudation. All these data enhance the current scarce knowledge of skin mucus as a target through which to study environmental changes and fish status.

Evaluating mucus exudation dynamics through isotopic enrichment and turnover of skin mucus fractions in a marine fish model.

Fish skin mucus is composed of insoluble components, which form the physical barrier, and soluble components, which are key for interrelationship functions. Mucus is continuously secreted, but rates of production and exudation are still unknown, as are the underlying mechanisms. Using stable isotope analysis, here, we evaluate skin mucus turnover and renewal in gilthead sea bream, separating raw mucus and its soluble and insoluble fractions. Isotopic abundance analysis reveals no differences between mucus and white muscle, thus confirming mucus samples as reliable non-invasive biomarkers. Mucus production was evaluated using a single labelled meal packaged in a gelatine capsule, with both 13C and 15N, via a time-course trial. 13C was gradually allocated to skin mucus fractions over the first 12 h and was significantly (4-fold) higher in the soluble fraction, indicating a higher turnover of soluble mucus components that are continuously produced and supplied. 15N was also gradually allocated to mucus, indicating incorporation of new proteins containing the labelled dietary amino acids, but with no differences between fractions. When existent mucus was removed, dietary stable isotopes revealed stimulated mucus neoformation dependent on the components. All this is novel knowledge concerning skin mucus dynamics and turnover in fish and could offer interesting non-invasive approaches to the use of skin mucus production in ecological or applied biological studies such as climate change effects, human impact, alterations in trophic networks or habitat degradation, especially of wild-captured species or protected species.

Skin Multi-Omics-Based Interactome Analysis: Integrating the Tissue and Mucus Exuded Layer for a Comprehensive Understanding of the Teleost Mucosa Functionality as Model of Study.

From a general structural perspective, a mucosal tissue is constituted by two main matrices: the tissue and the secreted mucus. Jointly, they fulfill a wide range of functions including the protection of the epithelial layer. In this study, we simultaneously analyzed the epithelial tissue and the secreted mucus response using a holistic interactome-based multi-omics approach. The effect of the gilthead sea bream (Sparus aurata) skin mucosa to a dietary inclusion of spray-dried porcine plasma (SDPP) was evaluated. The epithelial skin microarrays-based transcriptome data showed 194 differentially expressed genes, meanwhile the exuded mucus proteome analysis 35 differentially synthesized proteins. Separately, the skin transcripteractome revealed an expression profile that favored biological mechanisms associated to gene expression, biogenesis, vesicle function, protein transport and localization to the membrane. Mucus proteome showed an enhanced protective role with putatively higher antioxidant and antimicrobial properties. The integrated skin mucosa multi-interactome analysis evidenced the interrelationship and synergy between the metabolism and the exuded mucus functions improving specifically the tissue development, innate defenses, and environment recognition. Histologically, the skin increased in thickness and in number of mucous cells. A positive impact on animal performance, growth and feed efficiency was also registered. Collectively, the results suggest an intimate crosstalk between skin tissue and its exuded mucus in response to the nutritional stimulus (SDPP supplementation) that favors the stimulation of cell protein turnover and the activation of the exudation machinery in the skin mucosa. Thus, the multi-omics-based interactome analysis provides a comprehensive understanding of the biological context of response that takes place in a mucosal tissue. In perspective, this strategy is applicable for evaluating the effect of any experimental variable on any mucosal tissue functionality, including the benefits this assessment may provide on the study of the mammalian mucosa.

Proteomic characterization of primary cultured myocytes in a fish model at different myogenesis stages.

Myogenesis is a complex two-phase process of proliferation and differentiation, which seems to be greatly conserved in vertebrates. For the first time in fish, we identify the changes that occur in the proteome during this process in a gilthead sea bream (Sparus aurata) myocyte primary cell culture (on days 4, 8 and 12), using 2-D gel electrophoresis and LC-MS/MS. A significant increase of myogenin expression at day 8 marked the transition from proliferation to differentiation. Of the 898 spots in the proteome analysis, the 25 protein spots overexpressed on day 4 and the 15 protein spots overexpressed on day 8 indicate the end of proliferation and the beginning of differentiation, respectively. Proliferation was characterized by enrichment of proteins involved in actin cytoskeleton remodelling and in cellular metabolic processes (transcription, ubiquitination, response to stress and glucose metabolism). During differentiation, 41 proteins were overexpressed and 51 underexpressed; many of them related to biosynthetic processes (RNA and protein synthesis and folding, and pentose pathways), terminal myotube formation and muscle contraction. The main cellular processes of both phases of muscle development in fish are similar with those observed in mammals but extended in time, allowing sequential studies of myogenesis.

Oxidative attack during temperature fluctuation challenge compromises liver protein homeostasis of a temperate fish model.

Seasonal variations in water temperature are a natural stressor of temperate fish that affect growth performance and metabolism globally. Gilthead sea bream is one of the most economically interesting species in the Mediterranean; but its liver metabolism is affected by the cold season. However, the effects of cold on protein turnover mechanisms have hardly been studied. Here, we study the relationship between liver oxidative status and protein homeostasis pathways during a 50-day low temperature period at 14 °C, and subsequent recovery at two times: 7 days (early recovery) and 30 days (late recovery). Liver redox status was determined by measuring oxidised lipids and proteins, the glutathione redox cycle and major antioxidant enzymes activities. Protein turnover was analysed via liver protein expression of HSP70 and HSP90; proteasome 26S subunits and polyubiquitination, as markers of the ubiquitin-proteasome system (UPS); and cathepsin D, as a lysosomal protease. Low temperature exposure depressed antioxidant enzyme activities, affecting the glutathione redox cycle and reducing total glutathione levels. Both the UPS and lysosomal pathways were also depressed and consequently, oxidised protein accumulated in liver. Interestingly, both protein oxidation and polyubiquitination tagging depended on protein molecular weight. Despite all these alterations, temperature recovery reverted most consequences of the cold at different rates: with delayed recovery of total glutathione levels and oxidised protein degradation with respect to enzyme activities recovery. All these findings demonstrate that protein liver homeostasis is compromised after chronic cold exposure and could be the cause of liver affectations reported in aquaculture of temperate fish.

Comparison of several non-specific skin mucus immune defences in three piscine species of aquaculture interest.

Fish skin mucus is a viscous and semipermeable barrier made mainly of water, glycoproteins and soluble proteins. It represents an important defence against the environment and previous studies have reported the presence of different substances involved in immune defence responses in it. The aim of the present work was to characterize skin mucus protease activity by zymography and esterase activity of the subfamily of carboxylesterases in three species of interest for aquaculture: gilthead sea bream, sea bass and meagre. Mucus antioxidant power was also determined by adapting ferric reducing antioxidant power (FRAP) analysis. As a result of these non-specific immune defence parameters, we compared the antibacterial capacity of skin mucus in these species via in vitro dual bacteria strains-skin mucus co-culture growths. We used Pseudomonas anguilliseptica and Vibrio anguillarum as marine pathogenic bacteria and Escherichia coli as non-pathogenic. For each fish species, in the respective zymograms, we determined a pattern of proteolytic digestion bands. A high-molecular-weight band (around 200 kDa; H-band) was evident in sea bream and sea bass, and showed chymotrypsin activity. One or two intermediate-molecular-weight bands (around 75 kDa; I-bands) with non-trypsin and non-chymotrypsin activity, and putatively with metalloprotease activity, were evident in all species. Finally, low-molecular-weight bands (between 14 and 30 kDa; L-bands) showed distinct patterns for each species and matched trypsin activity. Despite the conservative pattern of digestion bands, the levels of total proteolytic activity (TPA) were 5 and 10 times higher in meagre than in sea bass and sea bream, respectively. In parallel, three carboxylesterase activities were detected in the mucus of the three fish species, using myristate (pNPM-CE activity), butyrate (pNPB-CE activity) and acetate (pNPA-CE activity) as substrates. Both pNPB-CE and pNPA-CE were the most abundant in fish mucus, and meagre was again the species with the highest levels. In contrast, the antioxidant power of meagre skin mucus was the lowest. We established the capacity of skin mucus to block or limit bacterial growth (lytic activity) using 24 h growth curves. The log-growth phase of V. anguillarum was strongly blocked by sea bream and meagre mucus for a few hours; but not by sea bass mucus. However, if mucus was not renewed, log-growth was at the end of 24 h studied period. For its part, P. anguilliseptica growth curve was delayed by the three mucus types during the entire growth period. Only meagre achieved lytic activity against E. coli growth. All parameters studied here will be of a great interest as non-invasive bioindicators of non-specific immune defences in fish skin mucus.

Using stable isotope analysis to study skin mucus exudation and renewal in fish.

Fish skin mucus is proposed as a novel target for the study of physiological condition and to conduct minimally invasive monitoring of fish. Whereas mucus composition has been a major interest of recent studies, no practical techniques have been proposed to gain understanding of the capacity and rhythm of production and exudation. Here, we used stable isotope analysis (SIA) with a labelled meal, packaged in gelatine capsules, to evaluate mucus production and renewal in a fish model, the gilthead sea bream (Sparus aurata). Mucus 13C- and 15N-enrichment reached higher levels at 12 h post-ingestion without significant differences at 24 h. When the formation of new mucus was induced, 13C-enrichment in the new mucus doubled whereas 15N-enrichment only increased by 10%. These results indicate the feasibility of adopting SIA in mucus studies and allow us to propose this methodology as a means to improve knowledge of mucus turnover in fish and other animals.

Redox Challenge in a Cultured Temperate Marine Species During Low Temperature and Temperature Recovery.

Aquaculture is a growing industry that is increasingly providing a sizable proportion of fishery products for human consumption. Dietary energy and temperature fluctuations affect fish health and may even trigger mortality, causing great losses in fish production during winter. To better understand this unproductive winter period in aquaculture, the redox status in a cultured marine species, the gilthead sea bream, was analyzed for the first time by inducing controlled temperature fluctuations and reducing dietary lipid content. Two groups of fish (by triplicate), differing in their dietary lipid content (18% vs. 14%), were subjected to 30 days at 22°C (Pre-Cold), 50 days at 14°C (Cold) and then 35 days at 22°C (Recovery). Plasma and liver redox metabolites (oxidized lipid, oxidized protein and thiol groups), liver glutathione forms (total, oxidized and reduced) and liver antioxidant enzyme activities were measured. Reducing dietary lipid content did not affect gilthead sea bream growth, glutathione levels or enzyme activities, but did reduce the amount of oxidized lipids. A sustained low temperature of 14°C showed a lack of adaptation of antioxidant enzyme activities, mainly catalase and glutathione reductase, which subsequently affected the glutathione redox cycle and caused an acute reduction in total hepatic glutathione levels, irrespective of diet. Antioxidant enzyme activities were gradually restored to their pre-cold levels, but the glutathione redox cycle was not restored to its pre-cold values during the recovery period used. Moreover, the lower lipid diet was associated with transiently increased liver oxidized protein levels. Thus, we propose that fish should be fed a low lipid diet during pre-cold and cold periods, which would reduce oxidized lipid levels without affecting fish growth, and a higher energy diet during the recovery period. Moreover, diets supplemented with antioxidants should be considered, especially during temperature recovery.

Skin mucus metabolites in response to physiological challenges: A valuable non-invasive method to study teleost marine species.

Knowledge concerning the health and welfare of fish is important to conserve species diversity. Fish mucosal surfaces, and particularly the skin, are of utmost importance to protect the integrity and homeostasis of the body and to prevent skin infections by pathogens. We performed three trials simulating different environmental and anthropogenic challenges: fish capture (air exposure), bacterial infection and fasting, with the aim of evaluating epidermal mucus as a non-invasive target of studies in fish. In this initial approach, we selected three well-known marine species: meagre (Argyrosomus regius), European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) for our study. Mucus viscosity was measured in order to determine its rheological properties, and mucus metabolite (glucose, lactate, protein and cortisol) levels were analysed to establish their suitability as potential biomarkers. Skin mucus appeared as a viscous fluid exhibiting clearly non-Newtonian behaviour, with its viscosity being dependent on shear rate. The highest viscosity (p < 0.05) was observed in sea bream. Mucus metabolites composition responded to the different challenges. In particular, glucose increased significantly due to the air exposure challenge in meagre; and it decreased during food deprivation in sea bream by a half (p < 0.05). In contrast, mucus protein only decreased significantly after pathogenic bacterial infection in sea bass. In addition, mucus lactate immediately reflected changes closely related to an anaerobic condition; whereas cortisol was only modified by air exposure, doubling its mucus concentration (p < 0.05). The data provided herein demonstrate that mucus metabolites can be considered as good non-invasive biomarkers for evaluating fish physiological responses; with the glucose/protein ratio being the most valuable and reliable parameter. Determining these skin mucus metabolites and ratios will be very useful when studying the condition of critically threatened species whose conservation status prohibits the killing of specimens.

Chronic Cold Stress Alters the Skin Mucus Interactome in a Temperate Fish Model.

Temperate fish are particularly sensitive to low temperatures, especially in the northern Mediterranean area, where the cold season decreases fish-farm production and affects fish health. Recent studies have suggested that the skin mucus participates in overall fish defense and welfare, and therefore propose it as a target for non-invasive studies of fish status. Here, we determine the mucus interactome of differentially expressed proteins in a temperate fish model, gilthead sea bream (Sparus aurata), after chronic exposure to low temperatures (7 weeks at 14°C). The differentially expressed proteins were obtained by 2D-PAGE of mucus soluble proteins and further assessed by STRING analyses of the functional interactome based on protein-protein interactions. Complementarily, we determined mucus metabolites, glucose, and protein, as well as enzymes involved in innate defense mechanisms, such as total protease and esterase. The cold mucus interactome revealed the presence of several subsets of proteins corresponding to Gene Ontology groups. "Response to stress" formed the central core of the cold interactome, with up-regulation of proteins, such as heat shock proteins (HSPs) and transferrin; and down-regulation of proteins with metabolic activity. In accordance with the low temperatures, all proteins clustered in the "Single-organism metabolic process" group were down-regulated in response to cold, evidencing depressed skin metabolism. An interactome subset of "Interspecies interaction between species" grouped together several up-regulated mucus proteins that participate in bacterial adhesion, colonization, and entry, such as HSP70, lectin-2, ribosomal proteins, and cytokeratin-8, septin, and plakins. Furthermore, cold mucus showed lower levels of soluble glucose and no adaptation response in total protease or esterase activity. Using zymography, we detected the up-regulation of metalloprotease-like activity, together with a number of fragments or cleaved keratin forms which may present antimicrobial activity. All these results evidence a partial loss of mucus functionality under chronic exposure to low temperatures which would affect fish welfare during the natural cold season under farm conditions.

Skin mucus proteome of gilthead sea bream: A non-invasive method to screen for welfare indicators.

In teleosts, the skin mucus is the first physical barrier against physical and chemical attacks. It contains components related to metabolism, environmental influences and nutritional status. Here, we study mucus and composition based on a proteome map of soluble epidermal mucus proteins obtained by 2D-electrophoresis in gilthead sea bream, Sparus aurata. Over 1300 spots were recorded and the 100 most abundant were further analysed by LC-MS/MS and identified by database retrieval; we also established the related specific biological processes by Gene Ontology enrichment. Sixty-two different proteins were identified and classified in 12 GO-groups and into three main functions: structural, metabolic and protection-related. Several of the proteins can be used as targets to determine fish physiological status: actins and keratins, and especially their catabolic products, in the structural functional group; glycolytic enzymes and ubiquitin/proteasome-related proteins in the metabolic functional group; and heat shock proteins, transferrin and hemopexins, in the protection-related group. This study analyses fish mucus, a potential non-invasive tool for characterising fish status, beyond defence capacities, and we postulate some putative candidates for future studies along similar lines.