----------------- INFORMACIÓN GENERAL ------------------- 1. Título del dataset Temporal dynamics of inflammation in gilthead seabream (Sparus aurata) induced by λ-carrageenan, LPS, and Poly I:C: From behaviour to gene expression 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 de Murcia Correo electrónico: josecarlos.campos@um.es ORCID: 0000-0003-0303-5412 Nombre: Maria José Cabrera-Álvarez Institución: Center of Marine Sciences (CCMAR/CIMAR LA), Faro 8005-226, Portugal Correo electrónico: maria@fishethogroup.net ORCID: 0000-0001-5646-8743 Nombre: Ana Rita Oliveira Institución: Center of Marine Sciences (CCMAR/CIMAR LA), Faro 8005-226, Portugal Correo electrónico: ana@fishethogroup.net ORCID: 0000-0003-2246-1513 Nombre: Gonçalo Duarte Oliveira Institución: Center of Marine Sciences (CCMAR/CIMAR LA), Faro 8005-226, Portugal Correo electrónico: goncalo@fishethogroup.net 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 Nombre: Florbela Soares Institución: IPMA, Portuguese Institute for the Ocean and Atmosphere/EPPO, Aquaculture Research Station of Olhão, Portugal Correo electrónico: fsoares@ipma.pt ORCID: 0000-0001-6075-2472 Nombre: João L. Saraiva  Institución: Center of Marine Sciences (CCMAR/CIMAR LA), Faro 8005-226, Portugal Correo electrónico: jsaraiva@ualg.pt ORCID: 0000-0002-8891-8881 3. Fecha de recogida de los datos (fecha única o rango de fechas): [14-03-2023---15-08-2024] 4. Fecha de depósito de los datos: [11-07-2024] 5. Idioma del conjunto de datos: Inglés ------------------------ INFORMACIÓN METODOLÓGICA ------------------------ 1.Descripción de la metodología utilizada para generar el conjunto de datos. 2. Material and methods 2.1. Animals One hundred and twenty juveniles (59.94 ± 1.04 g, 15.26 ± 0.10 cm) of the seawater teleost gilthead seabream were obtained and maintained from broodstock at the Estação Piloto de Piscicultura de Olhão of the Instituto Português do Mar e da Atmosfera (EPPO-IPMA, Portugal). The experiment was conducted in March 2023, in an open circulation system adjacent to the Ria Formosa lagoon, with a rate flow of 800 L h−1 and in which the sea water was filtered through sand filters. The tanks were located outdoors and were equipped with an artificial aeration system. They were shaded with dark mesh covers but were subjected to natural photoperiod and temperature fluctuations. The water temperature, salinity and dissolved oxygen were measured daily, yielding average values of 16.3 ± 1 ºC, 37.5–38 PSU, and 93.9 ± 0.4% O₂Sat, respectively. The fish were fed ad-libitum with commercial 6 mm pellets (Aller Blue Ex Vitamax by ALLER AQUA®, Denmark), specifically manufactured for gilthead seabream. The trials were conducted at EPPO-IPMA facilities, after compliance with internal ethics boards and under ethical permit 2023DGV/ 000066293 issued by Direção Geral de Alimentação e Veterinária (DGAV), Ministério da Agricultura, Florestas e Desenvolvimento Rural, Portugal in compliance with the European (Directive 2010/63/EU) and Portuguese (Decreto-Lei no.113/2013 de 7 de Agosto) legislation for the use of laboratory animals. All procedures were conducted by trained scientists under Group-C licences issued by DGAV, and under the supervision of an in-house veterinarian. 2.2. Experimental design and behaviour monitoring Fish were randomly selected, distributed to eight 300 L cylindrical fibreglass tanks, and acclimated to the tanks and the recording cameras (GoPro Hero 7®) for two weeks. Prior to the onset of the experiment, fish were filmed from the bottom of the tank for 20 min, to record their basal behaviour (Time 0). Then, fish were anesthetized with a bath of 2-phenoxyethanol (500 ppm, Sigma-Aldrich), and intramuscularly injected in the left flank, below the lateral line at the level of the second dorsal fin. Four groups of fish were established and the fish were injected with: i) 50 μL of phosphate-saline buffer (PBS, control group); ii) 50 μL of λ-carrageenin (0.5 mg fish-1 in PBS, Sigma, CAS Number: 9064-57-7); iii) 50 μL of Lipopolysaccharide from Escherichia coli (LPS, 0.5 mg fish-1 in PBS, Sigma, CAS Number: 297-473-0); iv) 50 μL polyinosinic-polycytidylic acid sodium salt (Poly I:C, 0.5 mg fish-1 in PBS, Sigma, CAS Number P1530). Each experimental group comprised 30 fish, which were distributed between two tanks (two replicates, with 15 fish per tank). Fish were recorded again for 20 min at 3, 6 and 24 h post-injection (henceforth, p.i.) to assess potential changes in behaviour due to inflammation. The first 5 min of each recording session were considered the habituation period to the camera and were excluded from the behavioural analysis. Behavioural observations were conducted during the subsequent 5 min of the recording, with point-scan sampling performed every minute. Behavioural parameters were classified into five categories as described in Table 1: i) spatial distribution (inner, outer, lower and upper), ii) display (light and dark colour), iii) swimming activity (freezing, regular and agitated), iv) social interactions (chase, escape, bite, opercular movement and interaction with structures) and v) feeding behaviour (anticipation, foraging, eating and non-appetite behaviours). For each behaviour, the proportion of fish in view exhibiting that specific behaviour was scored at each time point. Following the initial acclimation and 5-min behavioural observation, the fish were fed, and behaviour was assessed for an additional 5 min using the same observational protocol. For spatial distribution, because upper and outer behaviours mirror the lower and inner behaviours respectively, they were considered redundant and therefore not analysed. To validate behaviours related to swimming activity and social interactions, a 10-seconds segment of video prior to each point scan was analysed and extrapolated to represent a one-minute observation window. Foraging behaviour was assessed both before and after feeding, whereas anticipation and eating were specifically quantified during feeding events. All video recordings were analysed blind, with the video identities revealed only after the completion of data collection. 2.2. Sample collection Following the recordings at 3, 6 and 24 h p.i., three fish from each tank (n = 6 per group) were sedated as previously described and weighed. The diameter of the injection site was measured to assess any increase. Blood samples were then collected, and plasma was extracted by centrifuging the samples (10,000 x g, 5 min, 4 °C) and stored at -20 ºC until further analysis. Skin samples were obtained by dissecting the fish, rapidly frozen in liquid nitrogen, and stored at -80 ºC until utilised for gene expression analysis. 2.3. Stress parameters Plasma cortisol levels were measured by means of a commercial enzyme-linked immunosorbent assay (ELISA) kit (Tecan®, IBL International). Glucose and lactate levels in plasma were evaluated using commercial kits (Spinreact®) and following the manufacturer's procedures. 2.4. Gene expression by real-time PCR. The selected genes are enumerated in Table 2. Their sequences were obtained from a gilthead seabream database [80]. The Open Reading Frames (ORF) were located using the ExPASy translation software (SIB Bioinformatics Resource Portal) and an additional check was performed using NCBI BLAST sequence alignment analysis (NIH). The primers were designed with the Thermo Fisher OligoPerfectTM tool, according to the following criteria: i) each individual oligonucleotide was composed of 20 nucleotides, ii) the size of the amplicon had between 100 and 120 nucleotides, iii) with a guanidine-cytosine (GC)% between 55 % and 60 %, iv) a semi-naturalization temperature (Melting temperature) as close as possible to 60 °C, and v) the selection of primers that self-inhibit forming hairpins was avoided as far as it was possible, in order not to hinder the amplification reaction. The primers used are presented in Table 2. Total RNA was extracted from samples of 0.5 g of skin using TRIzol Reagent [81]. The extraction of RNA was performed following the manufacturer instructions and the quantification and purification was assessed by using the Nanodrop® spectrophotometer; 260:280 ratios were 1.8-2.0. Then, the RNA was treated with DNase I (Promega) to remove genomic DNA contamination and complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the SuperScriptIV reverse transcriptase enzyme (Life Technologies) with an oligo-dT18 primer. The expression of the selected genes (see Table 2) was analysed by real-time qPCR with QuantStudio™ Real-Time PCR System Fast (Life Technologies). Reaction mixtures [containing 5 µL of SYBR Green supermix, 2.5 µL of primers (0.6 µM each) and 2.5 µL of cDNA template] were incubated for 10 min at 95 ºC, followed by 45 cycles of 15 s at 95 ºC, 1 min at 60 ºC, and finally 15 s at 95 ºC, 1 min at 60 ºC and 15 s at 95 ºC. The gene expression was analysed using the 2−ΔCt method [82], which was performed as described elsewhere [83]. The specificity of the reactions was analysed using samples without cDNA as negative controls. For each mRNA, gene expression was normalized with the geometric mean of ribosomal protein (s18) and elongation factor 1-alfa (ef1a) expressions as house-keeping genes. Gene names follow the accepted nomenclature for zebrafish (http://zfin.org/). CT values lower than 40 were used for calculations. Samples in which the CT was undetermined the 2−ΔCt value for calculations was assumed as 0. 2.5. Data processing and Statistical analysis Behavioural data related to states (i.e., spatial distribution) reflect duration and were converted to proportions to account for differences in observation time, while all other behavioural data (i.e., social interactions), related to events and therefore being discrete and momentary, were analysed as frequencies to accurately capture how often these behaviours occur. For each behavioural parameter we used a linear mixed model (LMM) with sampling point (i.e., each of the 10 sampling observations within each time point) as the subject variable and random factor, time of sampling (i.e., 0, 3, 6, 24 h) as repeated measure and fixed factor, treatment as fixed factor, tank as a covariate, and each behavioural parameter as the dependent variable. We first ran an initial analysis including tank as fixed factor to check whether there was a potential tank effect on the behavioural data. Tank had no significant effect in all behavioural parameters except for opercular movement, so it was removed from the model for all behavioural parameters and left as a random factor, and the variable opercular movement was excluded from further analyses. We checked the normality of the residuals to meet the assumptions of the model. When there were significant effects, we ran pairwise comparisons with Sidak correction for multiple comparisons. When there were interactions between time and treatment, we ran two-way ANOVA pair-wise comparisons with Bonferroni corrections. Physiological data were analysed using a two-way ANOVA to identify significant differences between two factors: treatment and time, as well as to evaluate the interaction between these factors. Data normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was verified with Levene’s test. Outliers were identified and excluded during data analysis based on the interquartile range (IQR) criterion, where values falling below Q1 - 1.5 x IQR or above Q3 + 1.5 x IQR were removed (one in the glucose and three in the cortisol analyses). Results were expressed as mean ± standard error of the mean (SEM). All statistical analyses were performed with the SPSS software package (versions 28.0 and 29.0; IBM SPSS Statistics) for Windows. The significance level used was p < 0.05 for all statistical tests. 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: Computer package SPSS (28.0 version; SPSS Inc., Chicago, IL, USA) for Windows. 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]. Inflamafish 1 - Behaviour_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.]