----------------- INFORMACIÓN GENERAL ------------------- 1. Título del dataset Biomarker-based assessment of somatostatin-6 immunomodulation in gilthead Seabream: From serum proteins to skin mucus enzymes 2. Autoría: [Rellenar la información de todos los autores siguiendo el siguiente formato. Repetir el esquema, uno para cada autor.] Nombre: Jose Carlos Campos-Sánchez Institución: Universidad Miguel Hernández de Elche Correo electrónico: josecarlos.campos@um.es ORCID: 0000-0003-0303-5412 Nombre: Cristóbal Espinosa-Ruiz Institución: Instituto Murciano de Investigación y Desarrollo Agrario y Medioambiental (IMIDA) Correo electrónico: cristobal.espinosa@carm.es ORCID: 0000-0002-3063-1634 Nombre: Claudia Marín-Parra Institución: Universidad de Murcia Correo electrónico: claudia.marin@um.es ORCID: 0000-0002-7969-7052 Nombre: Francisco A. Guardiola Institución: Universidad de Murcia Correo electrónico: faguardiola@um.es ORCID: 0000-0002-1018-5446 Nombre: María Ángeles Esteban Institución: Universidad de Murcia Correo electrónico: aesteban@um.es ORCID: 0000-0002-6264-1458 3. Fecha de recogida de los datos (fecha única o rango de fechas): [10-11-2024---25-11-2024] 4. Fecha de depósito de los datos: [22-10-2025] 5. Idioma del conjunto de datos: Inglés ------------------------ INFORMACIÓN METODOLÓGICA ------------------------ 1.Descripción de la metodología utilizada para generar el conjunto de datos. 2. Materials and methods 2.1. Animals Thirty-six teleost gilthead seabream (S. aurata L.; mean weight: 52.2 ± 2.0 g; mean length: 14.0 ± 0.1 cm) were obtained from a local fish farm (Alicante, Spain). Fish were kept in recirculating seawater aquaria (450 L) in the Marine Fish Facilities at the University of Murcia (Spain) for a one-month quarantine prior to experimentation under the following conditions: water temperature was maintained at 20 ± 2 °C with a flow rate of 600 L h-1, salinity of 28 ‰, a 12 h light: 12 h dark artificial photoperiod, and continuous aeration. Water ammonium and nitrite levels were monitored in the tanks using specific kits (Seachem) and were maintained below the limits for the species (0.1 mg L-1 and 0.2 mg L-1, respectively). The fish were fed a commercial diet (Skretting) at 2 % body weight per day and fasted for 24 h before the trial. All procedures complied with the EU Directive 2010/63/EU and were approved by the UMU Ethics Committee (Permit No. A13160416). 2.2. Experimental design The fish were anesthetized with clove oil (20 mg L-1, Guinama®) and randomly assigned to one of the different experimental groups, depending on the intramuscular and intraperitoneal solutions administered. The intramuscular injection consisted of 500 µL of phosphate-buffered saline (PBS; 11.9 mM phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4, Fisher Bioreagents) or 1 % λ carrageenan (final dose = 10 mg fish-1, Sigma Aldrich) injected in their left flank beneath the lateral line at the level of the second dorsal fin. The intraperitoneal injection consisted of 100 µL of PBS or somatostatin 6 (SST6, GenScript Biotech). Six experimental groups were established: i) intramuscular  PBS +  intraperitoneal PBS (control); ii) intramuscular  λ carrageenan + intraperitoneal PBS (λ carrageenan); iii) intramuscular  PBS + 1 nM fish-1 SST6 intraperitoneal (SST6  1 nM); iv) intramuscular  PBS + 2 nM fish-1 SST6 intraperitoneal (SST6  2 nM), v) intramuscular  λ carrageenan + 1 nM fish-1 SST6 intraperitoneal (C+ SST6 1 nM), and vi) intramuscular  λ carrageenan + 2 nM fish-1 SST6 intraperitoneal (C+ SST6 2 nM). Each group was maintained in two replicate tanks (n = 3 fish per tank), resulting in six fish per treatment. Three days post-injection , the fish were anesthetized as described above, weighed, and measured. 2.3. Sample collection Skin mucus samples were collected by gently scraping the dorsolateral surface of the specimens using a cell scraper, taking care to avoid contamination with blood and/or urogenital and intestinal excretions [4]. The mucus samples were vigorously shaken and centrifuged (2,000 x g, 10 min, 4 ºC). The supernatants were stored at -80 ºC until use. Blood samples (200 μL) were extracted from the caudal vein using an insulin syringe. The samples were left to coagulate at 4 °C for 4 h, and the serum was collected after centrifuging the samples (10,000 x g, 5 min, 4 ºC) and stored at -20 ºC until use. Following blood collection, all fish were euthanized with an overdose of clove oil (100 mg L-1, Guinama®), ensuring that no fish were subjected to unnecessary stress or prolonged suffering after the experimental procedures. 2.4. Total protein and immunoglobulins concentration The protein concentration in each serum and skin mucus sample was determined using the Coomassie Brilliant Blue G-250 method [56]. Briefly, 5 μL of the samples in triplicate were incubated with 250 μL of Bradford reagent (Sigma-Aldrich) in 96-well flat-bottomed plates. After incubating the plates (10 min at room temperature in darkness), the absorbance at 595 nm was measured using a plate reader (BMG, FLUOstar Omega). The total protein concentration in each sample was expressed as mg mL-1. Total immunoglobulins (Ig) were determined based on the measurement of total protein content before and after precipitating the immunoglobulin molecules using a 12% solution of polyethylene glycol (PEG; Sigma-Aldrich) with 10 μL of serum or 50 μL of skin mucus samples [57]. Bovine serum albumin (BSA, Sigma-Aldrich) was used as the standard in both cases. The difference in protein content was considered to be the total immunoglobulin content in the plasma and skin mucus. 2.5. Electropherogram The proteinogram profile was determined as previously described [51]. Briefly, serum protein concentration was determined using an infrared-based protein quantitation method with a Direct Detect Spectrometer (Merck-Millipore). The serum samples were diluted 10 times in PBS, and 2 μL aliquots were spotted on the measuring card, with PBS used as the reference buffer. The measurements were performed using the standard bovine serum albumin calibration curve method. Based on the data obtained from infrared protein quantification, the serum samples were diluted in Milli-Q water and adjusted to 1,500 ng μL-1 concentration. Protein sizing and quantification were performed using the Protein230 kits on an Agilent 2100 Bioanalyzer (Agilent Technologies) according to the manufacturer’s instructions. Data analysis was performed using the Agilent 2100 Expert software. Size estimation compared peak migration times to an external protein standard (ladder) with predefined sizes ranging from 14 to 240 kDa, whereas concentration estimation compared peak areas to internal standard proteins (lower and upper markers, with 4.5 kDa and 240 kDa). 2.6. Humoral immune parameters 2.6.1. Natural haemolytic complement activity The activity of the alternative complement pathway in serum was assayed using pig red blood cells (pRBC) as targets [58]. Equal volumes of pRBC suspension (6%) in phenol red-free Hank's balanced salt solution (HBSS, Gibco) containing Mg+2 and ethylene glycol tetraacetic acid (EGTA) were mixed with serially diluted serum to obtain final serum concentrations ranging from 10% to 0.078%. After incubation for 90 min at 22 ºC, the samples were centrifuged (400 x g, 5 min, 4 ºC) to avoid unlysed erythrocytes. The relative hemoglobin content of the supernatants was assessed by measuring their optical density (OD) at 550 nm using a plate reader. The maximum (100%) and minimum (spontaneous) hemolysis values were obtained by adding 100 μL of distilled water or HBSS to 100 μL of PRBC samples, respectively. The degree of hemolysis (Y) was estimated, and the lysis curve for each specimen was obtained by plotting Y (1-Y)−1 against the volume of serum added (mL) on a log-log scaled graph. The volume of serum producing 50% hemolysis (ACH50) was determined, and the number of ACH50 units mL−1 was obtained for each experimental fish. 2.6.2. Peroxidase activity To determine the level of peroxidase activity, 5 µL of serum and 45 µL of skin mucus samples were added to PBS to a final volume of 50 µL in flat-bottomed 96-well plates. As substrates, 100 µL of 10 mM 3,3,5,5 tetramethylbenzidine hydrochloride (TMB; Sigma-Aldrich) and 10 µL of 0.015 % H2O2 were added. The reaction was stopped after 2 min by adding 50 µL of 2 M sulfuric acid (H2SO4), and the OD was read at 450 nm in a plate reader (BMG Labtech) [59]. Standard samples without serum or skin mucus were used as blank samples. One unit of peroxidase activity was defined as the amount producing an absorbance change of 1 OD, and the activity was expressed as U mL-1. 2.6.3. Esterase activity Esterase activity in serum and skin mucus samples was determined according to a previous protocol with slight modifications [26]. For mucus samples, equal volumes (100 µL) of sample and 0.4 mM p-nitrophenyl myristate substrate in 100 mM ammonium bicarbonate buffer containing 0.5% Triton X-100 (pH 7.8, 30 ºC) were mixed. In the case of serum, 10 µL of the sample was first diluted in 90 µL of the same ammonium bicarbonate buffer before adding the substrate solution. OD was continuously measured at 1 min intervals for 1 h at 405 nm using a plate reader. Standard samples without serum or skin mucus were used as blanks. The initial reaction rate was used to calculate the activity because the reaction followed a linear relationship. The activity was expressed as U mL-1, which was defined as the amount of enzyme required to release 1 µmol of p-nitrophenyl myristate in 1 min. 2.6.4. Lysozyme activity Lysozyme activity was measured using a turbidimetric method [60]. Briefly, 20 μL of serum or skin mucus samples were placed in 96-well plates. To each well, 180 µL of freeze-dried Micrococcus lysodeikticus (0.2 mg mL-1, Sigma) in 40 mM sodium phosphate buffer (pH 6.2) was added as a substrate. For blanks, 20 μL of serum or skin mucus were added to 180 μL of sodium phosphate buffer. The absorbance at 450 nm was measured after 20 min at 35 ºC using a microplate reader (BMG Labtech). Lysozyme levels in serum and skin mucus were obtained from a standard curve made with hen egg white lysozyme (HEWL, Sigma) through serial dilutions in the buffer. Lysozyme values are expressed as μg mL-1 equivalent to HEWL activity. 2.6.5. Protease activity Protease activity was quantified using the azocasein hydrolysis assay, as described previously [26]. Serum (20 μL) or skin mucus (100 μL) samples were incubated for 24 h at 22 ºC with 100 mM ammonium bicarbonate buffer containing 2% azocasein (Sigma). The reactions were stopped by adding 250 μL of 10% TCA for 30 min at room temperature and centrifuged (14,000 x g, 5 min). Supernatants were transferred to a 96-well plate in triplicate with 100 µL well-1 of 1 N NaOH. The OD was measured at 450 nm using a plate reader. Serum or skin mucus was replaced with trypsin solution (5 mg mL-1, Sigma) as a positive control or buffer as a negative control. The percentage of trypsin activity was then calculated. 2.6.6. Antiprotease activity Total antiprotease activity was determined by the ability of serum or skin mucus to inhibit trypsin activity with some modifications [61]. Briefly, aliquots of 10 μL of serum or 50 μL of skin mucus were incubated (10 min, 22 ºC) with the same volume of standard trypsin solution (5 mg mL-1). Subsequently, 100 µL of ammonium bicarbonate buffer and 125 µL of ammonium bicarbonate buffer containing 2% azocasein (Sigma) were added to the serum and skin mucus samples and incubated (120 min, RT). Finally, 250 µL of 10% trichloroacetic acid (TCA) was added, and a new incubation (30 min, RT) was carried out. The mixture was then centrifuged (10,000 x g, 5 min), and the supernatants were transferred to a 96-well plate in triplicate containing 100 µL well-1 of 1 N NaOH. OD was measured at 450 nm using a microplate reader (BMG Labtech). For the negative control (blank), the buffer replaced the serum or skin mucus (100% protease and 0% antiprotease activity), and for the positive control (reference sample), the buffer replaced the trypsin (0% protease and 100% antiprotease activity). The percentage of trypsin activity inhibition in each sample was calculated. 2.7. Data processing and statistical analyses. The protein concentration data obtained from the electropherograms were classified according to molecular weight. Proteins with a single peak in the electropherogram were directly averaged across individuals. Proteins with two or more peaks in an individual were summed, and the mean was calculated. The final data were obtained by taking the sum of all concentrations as 100 % and then adjusting the values accordingly. The results are expressed as the mean ± standard error of the mean (SEM). Data were analysed using one-way ANOVA followed by Tukey's post hoc tests to determine differences between experimental groups. Prior to analysis, the normality of the data was assessed using the Shapiro-Wilk test, and homogeneity of variance was verified using the Levene test. When the data did not meet parametric assumptions, a non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used. All statistical analyses were performed using RStudio V4.4.2 [62]. Pairwise Pearson correlation coefficients were calculated among all systemic and local humoral biomarkers using the rcorr() function from the Hmisc package. Correlation plots were generated using the corrplot package [63] to visualize the strength and direction of the associations. Only strong correlations (|r| ≥ 0.7) with statistical significance (p < 0.05) were considered relevant for interpretation. The results were exported as a formatted correlation matrix and as a long table, including the r and p values for each biomarker pair. The level of significance was set at P < 0.05 for all statistical tests. Figures were prepared using GraphPad Prism 8 software and RStudio. 2. Software o instrumentos necesarios para interpretar los datos: [Incluir la versión del software. Si hace falta un software específico de acceso restringido, explicar cómo obtenerlo. Valorar si es posible cambiar el conjunto de datos a un formato abierto (recomendado).] Data analysis: RStudio V4.4.2 3. Procedimientos seguidos para asegurar la calidad de los datos Positive and negative controls in each assay ------------------------ ESCTRUCTURA DE LOS ARCHIVOS --------------------------- 1. Nombres de archivos [Mencionar todos los archivos incluidos en el conjunto de datos, con el nombre y la extensión (.csv, .pdf, etc.) de cada fichero]. SST6 in vivo - Biomarcadores_Raw data.xls 2. Formato de los archivos: Libro de Excel 97-2003 (*.xls) ------------------------ MÁS INFORMACIÓN ------------------------ [Incluir cualquier otra información sobre el conjunto de datos que no haya quedado reflejada en esta plantilla y que se considere relevante.]