Caracterización funcional del mucus epidérmico en peces de interés productivo: Metodología no invasiva de identificación de moléculas biomarcadoras y relación con el estado fisiológico.

[spa] La acuicultura se ha convertido en una alternativa real y rentable a la pesca extractiva para proporcionar un alimento de un elevado valor nutritivo. Entre las especies de mayor relevancia en la producción acuícola marina del área Mediterránea, se encuentran la dorada (Sparus aurata), la lubina (Dicentrarchus labrax) y más recientemente la corvina (Argyrosomus regius). Si bien se han conseguido grandes adelantos en la mejora de la alimentación y el crecimiento, las metodologías más utilizadas de análisis para determinar su estado fisiológico son de carácter invasivo, ya sea por análisis sobre tejidos (lo que provoca la muerte del animal) o hematológicos (pueden provocar patologías adyacentes). Las superficies mucosas (en piel, branquias e intestino) de los peces teleósteos constituyen la primera línea de defensa contra la infección patógena además de participar en un amplio espectro de otros procesos fisiológicos muy importantes como la absorción de nutrientes, la osmoregulación, la respiración, la locomoción o la excreción de residuos. En la última década, el mucus epidérmico llegado a cautivar a muchos investigadores por su gran variedad de respuestas frente a diferentes condiciones y además, por tener un potencial muy importante para ser un método no invasivo de estudio del estado global del animal. Este trabajo se ha centrado en la característica no invasiva y potencial del mucus epidérmico. Por otro lado, tiene como objetivo principal obtener un conjunto completo de marcadores biológicos o biomarcadores (definidos en este trabajo como biomarcadores asociados al mucus de la piel o SMABs, por sus siglas en inglés “Skin Mucus Associated Biomarkers” del estado fisiológico en el mucus para poder establecer su relación con la respuesta a los cambios ambientales y nutricionales. Para ello, se propuso: caracterizar los componentes proteicos del mucus en dorada, estudiar la respuesta mediante diferentes agentes estresores, ya sean agudos o crónicos y caracterizar y comparar algunas de las propiedades defensivas del mucus entre las tres especies. Como resultado de todos estos objetivos, se han identificado una serie de posibles SMABs ordenados de la siguiente manera: 1) características físico-químicas, dónde se propone el estudio reológico del mucus como un posible informador de un estrés agudo; 2) metabolitos y hormonas, estudiando sus cambios en el mucus, sus niveles de exudación puede informar sobre respuestas agudas, como por ejemplo en los metabolitos como la glucosa, el lactato, la proteína o en la hormona cortisol; 3) enzimas con función defensiva no-específica, tanto proteasas como esterasas tienen un papel muy importante en el mucus, por ello se estudió el zimograma de la actividad proteasa y la familia de las carboxilesterasas, como posibles marcadores biológicos frente a un estrés crónico; y 4) otras moléculas principalmente de estrés crónico, como: la actina y sobretodo sus isoformas, importantes informadoras sobre la secreción del mucus; diferentes fragmentos de queratina, propuestos como péptidos antimicrobianos; moléculas relacionadas con el metabolismo de la glucosa, que pueden informar sobre el estado metabólico interno de los animales; moléculas relacionadas con el metabolismo proteico, importantes para determinar la estructura del mucus o activadoras de varios componentes; y finalmente moléculas con un carácter protector como las transferrinas o diferentes chaperonas. Con todo ello, finalmente se determinó también la actividad bactericida del mucus epidérmico como indicativo del potencial real defensivo contra posibles infecciones. En las conclusiones del trabajo se explican los beneficios de estudiar las propiedades y componentes del mucus de la piel como indicativos del estado del animal y de manera no invasiva, indicando qué componentes pueden ser más interesantes de analizar de manera rápida y qué componentes responden a desafíos agudos o crónicos.[eng] Aquaculture has become a real and profitable alternative to extractive fishing, providing food with high nutritional value. In this work, we select three marine species with a great interest in Mediterranean aquaculture: gilthead sea bream (Sparus aurata), sea bass (Dicentrarchus labrax) and meagre (Argyrosomus regius), with the purpose to evaluate the health and welfare status of the animals through non-invasive methods, selecting putative biomarkers. Our aim was to use skin mucus as it plays an important role in innate defense, but also in other physiological processes such as osmoregulation, respiration, locomotion or waste excretion. For that, fish were subjected to different stress factors (acute and chronic), and different techniques were used to reveal a wide range of biomarkers, also described in the present study as Skin Mucus Associated Biomarkers, SMABs. As result, we propose the following biological markers: 1) related to rheology, mucus viscosity was selected as acute stress indicator; 2) related to metabolism response, metabolites such as glucose, lactate or protein, together with exuded cortisol, its mucus levels could report on acute responses like hypoxia or netting, and chronic stressors like fasting periods in sea bream; 3) related to enzymatic activities, several protease types could be good biomarkers of fish defense status or mucus removal, mainly the metalloprotease activity detected at chronic low temperatures, or esterases subfamily of carboxylesterase, within which we detected at least three different activities in fish mucus, all of them related to detoxifying capacity to different toxics; 4) related to different soluble mucus proteins by proteasome studies, like some structural β-actin or keratin isoforms (KDAMPs), some metabolic proteins related to glucose and protein metabolism, or protective proteins with different functions like transferrin or Heat Shock Proteins (HSPs). Finally, we also determined the bactericidal activity of the epidermal mucus as an indication of the real defensive potential against possible infections. In the conclusions section, we explain the benefits of studying the properties and components of the skin mucus as indicative of the animal state non-invasively, indicating which components can be more interesting to analyze quickly and which components would better respond to acute challenges or chronic conditions

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

Multibiomarker approach to fipronil exposure in the fish Dicentrarchus labrax under two temperature regimes

Fipronil is a phenylpyrazole insecticide widely used to control pests in agriculture even though evidence of harmful side effects in non-target species has been reported. A comprehensive study on the effects of dietary administration of Regent®800WG (80 % fipronil) in European sea bass juveniles was carried out under two temperature regimes: a) natural conditions, and b) 3 °C above the natural temperature (an increase predicted for the NW Mediterranean by the end of this century). Fipronil was added to the fish food (10 mg fipronil /Kg feed) and the effects were studied at several time points including right before administration, 7 and 14 days after daily fipronil feed and one-week after the insecticide withdrawal from the diet (depuration period). A wide array of physiological and metabolic biomarkers including feeding rate, general condition indices, plasma and epidermal mucus metabolites, immune response, osmoregulation, detoxification and oxidative-stress markers and digestive enzymes were assessed. General linear models and principal component analyses indicated that regardless of water temperature, fipronil resulted in a significant alteration of several of the above listed biomarkers. Among them, glucose and lactate levels increased in plasma and decreased in epidermal mucus as indicators of a stress response. Similarly, a depletion in catalase activity and higher lipid peroxidation in liver of fipronil-exposed fish were also indicative of an oxidative-stress condition. Fipronil induced a time dependent inhibition of Cytochrome P450-related activities and an increase of phase II glutathione-S-transferase. Moreover, fipronil administration was able to reduce the hypo-osmoregulatory capability as shown by the increase of plasmatic osmolality and altered several digestive enzymes including trypsin, lipase, alpha amylase and maltase. Finally, analyses in bile and muscle confirmed the rapid clearance of fipronil but the persistence of the metabolite fipronil-sulfone in bile even after the 7-day depuration period. Altogether, the results reveal a notable impact of this compound on the physiological condition of the European sea bass. The results should be considered in future environmental risk assessment studies since fipronil could be hazardous to fish species, particularly those inhabiting estuarine ecosystems exposed to the discharge of agriculture runoffs where this pesticide is mainly used

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

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

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

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

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

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-1β), and anti-inflammatory (tgf-β1, 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

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). 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-1β), and anti-inflammatory (tgf-β1) 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