----------------- INFORMACIÓN GENERAL ------------------- 1. Título del dataset Data set of Novel Hepcidin Genes in Gilthead Seabream: Implications for Immune Response and Iron Metabolism 2. Autoría: [Rellenar la información de todos los autores siguiendo el siguiente formato. Repetir el esquema, uno para cada autor.] Nombre: Laura García-Navarro Institución: Universidad de Murcia Correo electrónico: laura.garcia12@um.es ORCID: 0009-0007-6678-8426 Nombre: Jhon A. Serna‑Duque Institución: Universidad de Murcia Correo electrónico: jhonalberto.sernad@um.es ORCID: 0000-0001-6962-3722 Nombre: Alberto Cuesta Peñafiel Institución: Universidad de Murcia Correo electrónico: alcuestan@um.es ORCID: 0000-0002-0906-7995 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): [22-10-2023--01-05-2024] 4. Fecha de depósito de los datos: [24-04-2025] 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. Synteny and phylogeny analysis of hamp2.0 and hamp2.15 in the class Actinopterygii The genomic synteny analysis of hamp2.0 and hamp2.15 was conducted utilizing the platform https://www.genomicus.bio.ens.psl.eu/, visualized through Default View with Aliview mode. The investigation encompassed 129 fish genomes, with Actinopterygii (~386 Mya) selected as the common ancestor. These genome assemblies are accessible in Ensembl (Table S5). The phylogenetic tree was constructed employing the Neighbor-Joining method and the MEGA X program. The bootstrap test utilized 10,000 replicates [38]. For this analysis, hepcidin protein sequences from various species were utilized (Table S6). 2.3. Secondary structure prediction of HAMP2α or HAMP2Ω Secondary structure prediction of both sequences was performed using Prediction of the Secondary Structure by GOR (ocha.ac.jp) [39] and the Alphafold database on the https://benchling.com/ platform [40,41]. 2.2 Bactericidal activity of HAMP2α or HAMP2Ω Bactericidal activity was analyzed using two pathogens: V. harveyi (isolated from infected European sea bass, Dicentrarchus labrax) [43] and V. anguillarum. Additionally, the peptides’ bactericidal activity was tested against a probiotic from healthy gilthead seabream skin (Shewanella putrefaciens, SpPdp11) [44]. Bacteria were grown on Tryptic Soy Agar (TSA, Difco Laboratories) at 25 ºC, and then inoculated in liquid Tryptic Soy Broth (TSB, Difco Laboratories). Both media were supplemented with NaCl to a final concentration of 1.5 % (w v-1) [45]. The bacteria inoculated in TSB were incubated for 24 h at 25 ºC with shaking (200-250 rpm). The bacterial concentration was adjusted to 3.75 × 107 CFU ml-1 following determination of the optical density (OD) at 620 nm in relation to a standard curve [46,47]. Mature synthetic peptides of both hepcidins were synthesized using solid-phase peptide synthesis and subsequently procured (SynPeptide Co). Different PBS concentrations were tested to improve the solubility of both mature synthetic peptides. HAMP2α and HAMP2Ω were diluted in PBS 0.1 × and PBS 1.5 ×, respectively. For bactericidal activity, aliquots of 10 μL of bacteria and 10 μL of one of the mature synthetic hepcidin peptide (HAMP2α or HAMP2Ω) at different concentrations (0 μM, 3.12 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM) were added to each well of a 96-well flat-bottom plate. The samples were incubated (25 °C, 2 h, shaking at 200-250 rpm), after which the absorbance at 600 nm was measured every hour from 4 to 12 h of incubation. Percentage of bacterial inhibition was calculated using the formulae: 1- [(Optical density (OD) of Samples - OD of negative control) × (OD of positive control -OD of negative control)-1] × 100. Percentage of bacterial growth was calculated using the formulae: [(OD of Samples - OD of negative control) × (OD of positive control -OD of negative control)-1] × 100. All assays were performed in triplicate, and the results were expressed as a percentage. Additionally, TSB was utilized as a negative control, and the respective bacteria in TSB served as a positive control. 2.3. Cytotoxicity activity of HAMP2α or HAMP2Ω The cell line SAF-1 (ECACC - European Collection of Authenticated Cell Cultures- nº 00122301) was used in the assays. The cells were seeded in 25 cm2 plastic tissue culture flasks (Nunc, Germany) and cultured in L-15 Leibowitz medium (Sigma), supplemented with 10 % fetal bovine serum (FBS, Sigma), 2 mM L-glutamine, 100 international units (i.u.) mL-1 penicillin and streptomycin (Biowest) 100 mg mL−1. Cells were grown at 25 °C under humidified atmosphere (85 % humidity, 5 % CO2) (Heraeus BBK 6220). Exponentially growing cells SAF-1 monolayers were detached from culture flasks by 3 min exposure to 0.25 % of trypsin (Sigma) in phosphate-buffered saline (PBS), pH 7.2–7.4, according to the standard trypsinization methods. The detached cells were collected by centrifugation (200 g, 5 min, 25 °C) and cell viability was determined by trypan blue exclusion test. Cell concentration was determined by an automatic counting chamber (BioRad) and adjusted to 1.5 × 104 cells mL-1. Before being used in the assays, the mycoplasma test was performed according to the manufacturer's instructions (Thermo Fisher Scientific) to check that the cells were not contaminated. A MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay was performed. A 96-well microplate was inoculated with 100 μL of SAF-1 cell line (previously adjusted to 1.5 × 10⁴ cells well⁻¹) and incubated for 24 h (25 °C, 85 % humidity, 5 % CO₂). Subsequently, 100 μL of one of the two peptides (HAMP2α or HAMP2Ω) was added at various concentrations (0 μM, 6 μM, 12 μM, 25 μM, 50 μM or 100 μM), and the cells were incubated for an additional 24 h under identical conditions. The medium was then removed, and 200 μL of MTT (1 mg ml⁻¹, Sigma) was added. Following a 4-h incubation (25 °C, 5 % CO₂), the samples were centrifuged (2000 × g, 10 min) to remove the supernatant. Finally, 100 μL of dimethyl sulfoxide (DMSO, Sigma) were added, and absorbance measurements were obtained at 570 nm and 690 nm using a microplate reader [48]. The percentage of de cell viability was calculated with the formula: [((Optical density (OD) of Samples 570 nm - OD of negative control 570 nm) - (OD of samples 690 nm - OD of negative control 690 nm)) / ((OD of positive control 570 nm - OD of negative control 570 nm ) - (OD of positive control 690 nm - OD of negative control 690 nm ))] × 100. 2.4. Animals A local farm (Alicante, Spain) provided sixty healthy gilthead seabream specimens for the study (25 g average weight). The fish were housed in the University of Murcia's Marine Fish facilities, using 450-500 L seawater tanks. The water conditions were maintained at 28 ‰ salinity and 24 ± 2 ºC, with a 12-hour light-dark cycle. The specimens were given a commercial pelleted diet (Skretting) daily, amounting to 1 % of their body weight. Before experiments began, the fish underwent a 15-day quarantine period for acclimation. All procedures, including fish care, experimental protocols, and euthanasia, were conducted in accordance with ethical standards. The University of Murcia's Ethical Committee for Animal Experimentation approved the protocol (permit number A13150104), which adhered to the European directive 2010/63/EU concerning the protection of animals used for scientific purposes. 2.5. In vitro stimulation of head-kidney leucocytes A random selection of ten specimens was made, which were then euthanized using clove oil (Guinama) as an anesthetic and subsequently exsanguinated. Leucocytes were extracted from head kidney (HK) samples, while additional HK, spleen, and thymus samples were removed and promptly preserved in TRIzol reagent (Invitrogen) for later RNA extraction. For leucocyte obtention, HK was dissected and passed through cell filters (100 μm of pore size, Fisherbrand™) with 20 mL of L-15 medium (Leibovitz) to obtain head kidney leucocytes (HKLs). The culture media was previously supplemented with 0.35 % sodium chloride (to adjust the medium osmolality to gilthead seabream's plasma osmolality), 1% heparin (Biowest, to prevent coagulation), penicillin (to prevent contamination), L-glutamine and 3 % Fetal Calf Serum (FCS, Gibco). HKLs were centrifuged (485 x g, 10 min), resuspended and washed twice using L-15 supplemented medium without heparin. Using an automated cell counter, the cellular density was measured and subsequently adjusted to achieve a concentration of 107 cells mL-1. The viability of HKLs was evaluated by flow cytometry utilizing propidium iodide (PI, Sigma-Aldrich, a red fluorescent dye commonly employed to identify non-viable cells). Aliquots of 50 μL of PI solution (400 mg mL−1) were added to 5 mL Falcon tubes containing 100 μL of HKLs (n = 5) previously incubated for 0 h or 2 h with the gilthead seabream peptides HAMP2α or HAMP2Ω (GenScript) [at final concentrations of 0 (control), 12.5 and 25 µM]. Prior to analysis, the samples were gently mixed. Flow cytometry analysis was performed on a FACScan flow cytometer (Becton Dickinson) equipped with a 488 nm argon-ion laser and 10,000 cells were recorded per sample at a rate of 300 cells s-1. Data were acquired by two-parameter side scatter (SSC) and forward scatter (FSC). Graphical representations in the form of dot plots or histograms were generated for red fluorescence (FL2). For positive controls, 50 μL of trypan blue were added to lyse cells. Viable cells were calculated by subtracting 100 % from the estimated dead cells, which are the percentage of cells displaying PI (red) fluorescence. The pathogenic bacterium V. anguillarum R-82 was cultured for 24 h at 22 °C in tryptic soy broth (Conda Laboratories) supplemented with 1.5 % NaCl. Bacterial cell cultures were standardized by absorbance at 600 nm to 108 colonies forming units (c.f.u.) mL-1 and heat inactivated (60 ºC, 30 min). Nodaviruses (strain 411/96, genotype RGNNV) were propagated on the SSN-1 cell line (CVCL_4306) at 25 °C [49] and stocks were titrated in 96-well plates prior to utilization in experiments [50]. Aliquots of 107 HKLs mL-1 from five different fish specimens were incubated in 48-well flat-bottom microtiter plates (Nunc) with various stimuli which comprised: L-15 culture medium supplemented as previously described (control), lipopolysaccharide (LPS, 5 µg mL-1, Sigma), polyinosinic acid (poly I:C, 25 µg mL-1, Sigma), V. anguillarum (108 units forming colonies, UFC mL-1) or nodavirus (106 DICT50 mL-1). Following 24 h incubation at 25 °C, HKLs were washed and preserved in TRIzol reagent at -80 °C for subsequent RNA isolation. 2.6. In vivo stimulation of gilthead seabream hepcidins (hamp2.0 and hamp2.15) transcription Thirty-six gilthead seabream specimens were randomly selected, anesthetized with clove oil (20 mg L-1, Guinama®) and injected. Three groups of fish (with two replicates per group) were established. Bacterial challenge: fish were intramuscularly injected in the left flank, beneath the lateral line at the level of the second dorsal fin with 50 μL sterile PBS (control group) or with 108 UFC mL−1 heat-inactivated V. harveyi (bacteria group, obtained as described below) [36]. Four hours post-injection, the fish were dissected, and blood samples, as well as samples from the primary organ targets of the bacteria (brain, liver, spleen, foregut, HK, and skin), were collected [51]. Viral challenge: fish were intramuscularly injected with 100 μl of SSN-1 culture medium (control) or 100 μl of SSN-1 with 106 nodavirus DICT50 fish -1. One and 7 days after viral injection, HK (main hematopoietic organ) and brain (main replication organ of the virus) samples were collected [50]. Iron overload: fish were administered an intraperitoneal injection of 50 μL of sterile PBS as a control or 50 μL of 10 mg ml-1 iron dextran (Sigma-Aldrich) diluted in sterile PBS [52,53]. Liver (main organ for iron storage in fish) [54] and HK samples were obtained 72 h after injection. Following the development of the three in vivo assays, all acquired samples were immediately preserved in TRIzol Reagent (Invitrogen) and kept at -80 °C until needed for real-time PCR analysis. The extraction of total RNA from these frozen samples was conducted according to the manufacturer's protocol. Genomic DNA was eliminated from one microgram of total RNA using DNAse I (Invitrogen) treatment. Subsequently, the initial cDNA strand was produced through reverse transcription, employing ThermoScriptTM RNAse H− Reverse Transcriptase (Invitrogen) and an oligo-dT12–18 primer (Invitrogen), followed by RNAse H (Invitrogen) processing. A QuantStudio™ 5 Real-Time PCR System (Applied Biosystems) was utilized for fast qPCR, incorporating SYBR Green PCR Core Reagents (Applied Biosystems), cDNA samples, and custom-designed primers (Table S7). The reaction mixtures underwent incubation at 95 ºC for 10 min, then 40 cycles of 95 ºC for 15 s and 60 ºC for 1 min, concluding with 15 s at 95 ºC, 1 min at 60 ºC, and 15 s at 95 ºC. Melt curve analysis was performed on the PCR products. Hepcidin mRNA Ct values were standardized against elongation factor 1 alpha (ef1a) and ribosomal protein S18 (rp18S) housekeeping genes. The expression ratios of Vibrio-infected samples were compared to control cDNA using the Livak Method [55]. Primer design was performed with the Oligo Perfect software tool (Invitrogen), and the in silico specificity of all predicted potential primers was verified by Primer-BLAST using the complete gilthead seabream genome. Fast qPCR was performed with QuantStudio™ 5 Real-Time PCR, as above, with the identified specific primers and single cDNAs (Table S6). For experimental verification of primer specificity, qPCR products of the most concentrated HK and gut single cDNAs were used. The predicted sequences of the amplicons (Supplementary Fig 1A) were compared with the Sanger-sequenced products by pairwise alignment Melt curves of each amplicon were analyzed (Supplementary Fig. 1B). 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) RNA content in each sample. Gene names follow the accepted nomenclature for zebrafish (http://zfin.org/). In all cases, each PCR was performed on triplicate samples [55]. 2.7. Statistical study Statistical differences in the in vitro (HKLs stimulation, cytotoxic activity and bactericidal activity) and in vivo (nodavirus expression) assays were analyzed by one-way ANOVA (Prism7) or two-way ANOVA (Prism9), as required by the analysis. Statistical differences in hepcidin expression in in vivo samples (from fish stimulated by bacterial challenge and iron overload) were analyzed by t-student, Mann-Whitney test (Prism 7). For all statistical analyses, the significance level used was P < 0.05 and P-values are indicated as follows: *P < 0.05, **P < 0.01 and ***P < 0.001. 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).] RqPCR data: QuantStudio TM Design & Analysis Antimicrobial and Citotoxicity result: Microsoft Excel Sintenia (.svg): Inkscape SST-CST_-_Readme_Hamp2.0 and hamp2.15: Bloc de notas 3. Procedimientos seguidos para asegurar la calidad de los datos In all cases, negative controls and a satisfactory number of replicates were carried out to ensure the quality of the data. ------------------------ 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]. RqPCR.zip antimicrobial activity result.xlsx MTT_SAF_Hamp2.0 and Hamp2.15.xls Sintenia hamp2.15.ENSSAUG00010015977-Actinopterygii.svg Sintenia hamp2.0-ENSSAUG00010015954-Actinopterygii.svg SST-CST_-_Readme_Hamp2.0 and hamp2.15.txt 2. Formato de los archivos: Formatos: .xls .xlsx .zip .svg .txt ------------------------ MÁS INFORMACIÓN ------------------------ [Incluir cualquier otra información sobre el conjunto de datos que no haya quedado reflejada en esta plantilla y que se considere relevante.]