ORIGINAL RESEARCH published: 12 March 2021 doi: 10.3389/fimmu.2021.633621 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 Joana P. Firmino 1,2,3 , Laura Fernández-Alacid 4 , Eva Vallejos-Vidal 5 , Ricardo Salomón 1,3 , Ignasi Sanahuja 4 , Lluis Tort 6 , Antoni Ibarz 4 , Felipe E. Reyes-López 6,7,8* and Enric Gisbert 1* Edited by: 1 Benjamin Costas, IRTA, Centre de Sant Carles de la Ràpita (IRTA-SCR), Aquaculture Program, Sant Carles de la Ràpita, Spain, 2 University of Porto, Portugal TECNOVIT–FARMFAES, S.L. Pol. Ind. Les Sorts, Alforja, Spain, 3 Ph.D. Program in Aquaculture, Universitat Autònoma de Barcelona, Bellaterra, Spain, 4 Department of Cell Biology, Physiology and Immunology, Faculty of Biology, University of Reviewed by: Barcelona, Barcelona, Spain, 5 Departamento de Biología, Facultad de Química y Biología, Centro de Biotecnología Acuícola, Bernardo Baldisserotto, Universidad de Santiago de Chile, Santiago, Chile, 6 Department of Cell Biology, Physiology and Immunology, Universitat Federal University of Santa Autònoma de Barcelona, Bellaterra, Spain, 7 Facultad de Medicina Veterinaria y Agronomía, Universidad de Las Américas, Maria, Brazil Santiago, Chile, 8 Consorcio Tecnológico de Sanidad Acuícola, Ictio Biotechnologies S.A., Santiago, Chile Samuel A. M. Martin, University of Aberdeen, United Kingdom One of the main targets for the use of phytogenics in aquafeeds is the mucosal tissues as *Correspondence: they constitute a physical and biochemical shield against environmental and pathogenic Enric Gisbert Enric.Gisbert@irta.cat threats, comprising elements from both the innate and acquired immunity. In the present Felipe E. Reyes-López study, the modulation of the skin transcriptional immune response, the bacterial growth Felipe.Reyes@uab.cat 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 Specialty section: This article was submitted to thymol were assessed. The enrichment analysis of the skin transcriptional profile of fish Nutritional Immunology, fed the phytogenic-supplemented diet revealed the regulation of genes associated to a section of the journal Frontiers in Immunology cellular components involved in the secretory pathway, suggesting the stimulation, and Received: 25 November 2020 recruitment of phagocytic cells. Genes recognized by their involvement in non-specific Accepted: 11 February 2021 immune response were also identified in the analysis. The promotion of the secretion Published: 12 March 2021 of non-specific immune molecules into the skin mucus was proposed to be involved in Citation: the in vitro decreased growth capacity of pathogenic bacteria in the mucus of fish fed Firmino JP, Fernández-Alacid L, Vallejos-Vidal E, Salomón R, the phytogenic-supplemented diet. Although the mucus antioxidant capacity was not Sanahuja I, Tort L, Ibarz A, affected by the phytogenics supplementation, the regulation of genes coding for oxidative Reyes-López FE and Gisbert E (2021) Carvacrol, Thymol, and Garlic stress enzymes suggested the reduction of the skin oxidative stress. Additionally, the Essential Oil Promote Skin Innate decreased levels of cortisol in mucus indicated a reduction in the fish allostatic load due to Immunity in Gilthead Seabream the properties of the tested additive. Altogether, the dietary garlic, carvacrol, and thymol (Sparus aurata) Through the Multifactorial Modulation of the appear to promote the gilthead seabream skin innate immunity and the mucus protective Secretory Pathway and Enhancement capacity, decreasing its susceptibility to be colonized by pathogenic bacteria. of Mucus Protective Capacity. Front. Immunol. 12:633621. Keywords: SALT, innate immunity, stress, aquaculture, Vibrio infection, teleost fish skin mucus, phytogenic doi: 10.3389/fimmu.2021.633621 additive, interactome Frontiers in Immunology | www.frontiersin.org 1 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity INTRODUCTION garlic essential oil, carvacrol, and thymol (the main bioactive compounds of Labiateae plants essential oils) in a standard on- Fish infectious diseases are one of the main constraints of the growing diet for gilthead seabream (Sparus aurata), assessing aquaculture sector, representing a serious economic, social, and its effects on the skin transcriptional response, pathogenic environmental challenge for the industry (1). Since fish farmers bacterial growth capacity in skin mucus, and fish overall health depend on high survival rates and healthy animals, strategies condition. This species was chosen since it is the most important to improve their performance, immune status, and welfare are marine farmed fish species in the Mediterranean with an annual highly demanded for supporting health management practices production of 85,385.1 t in 2018 and an economic value of that positively impact in the final revenue of the fish farm. On 502,398,000 US$ (16); thus, improving health management this basis, the development of functional feed additives designed strategies based on sustainable dietary approaches for this farmed to physiologically support fish to cope with pathogenic and other species is of relevance for this growing industry. external challenges intrinsic to aquaculture rearing conditions, represents a promising tool to be implemented in a sustainable and environmentally-responsible aquaculture industry (2). MATERIALS AND METHODS Functional feeds containing essential oils, one of most Diets and Fish Rearing commonly used group of phytogenics in aquafeeds, have Juveniles of gilthead seabream were purchased from a received increased attention during these last years due to their Mediterranean fish farm (Piscicultura Marina Mediterránea antimicrobial, immunostimulant, antioxidant, anti-stress, and S.L., Andromeda Group, Valencia, Spain) and on-grown at growth-promoting properties (3–5). Besides essential oils being IRTA–Sant Carles de la Ràpita facilities for research purposes repeatedly demonstrated to stimulate both humoral and cellular (17). Before the onset of the trial, fish were individually measured components of the fish innate immunity (6), numerous have in body weight (BW) and standard length (SL) to the nearest been also shown to display a noteworthy antimicrobial activity 0.1 g and 1 mm, respectively (BW = 40.3 ± 0.1 g; SL = 12.0 ± against a wide range of fish pathogens (7, 8), putting them on 0.2 mm). Then, 150 juveniles were randomly distributed among the spotlight for the development of sustainable prophylactics. six 450 L tanks (25 fish per tank; initial density = 2 kg m−3 ; Particularly, phytogenics derived from garlic (Allium sativum L., three replicate tanks per experimental group) connected to an Alliaceae, Liliaceae), oregano (Oreganum vulgare, Labiateae), and IRTAmar R system working under an open-flow regime. thyme (Thymus vulgare, Labiateae) are among the most studied Fish were fed two experimental diets, one devoid of the and administrated, due to their recognized health-promoting functional feed additive (control diet) and a second one properties for aquatic species (6). supplemented with 0.5% of a microencapsulated functional One of the main targets for this type of nutritional additive containing synthetic garlic essential oil, carvacrol, and strategies is the mucosal tissues, due to their importance in the thymol (AROTEC-G R , TECNOVIT-FARMFAES, S.L., Spain). protection against the immediate contact with the environment Both diets were tested in triplicate tanks and administered and potential pathogens. Besides acting as a physical barrier, for a period of 65 days. Fish were hand-fed two times per the mucosal layer also offers a biochemical shield, in which day at the daily rate of 3.0% of the stocked biomass, which elements from both the innate and acquired immunity are approached apparent satiation. The control diet was formulated present (9). In particular, the fish skin mucus represents with high levels of marine-derived protein sources (30% fishmeal, the largest mucosal barrier with its whole epidermis directly 2.5% soluble protein concentrate—CPSP 90 R and 2.5 krill exposed to the environment. It is responsible for the first meal), containing 46% crude protein, 18% crude fat, and line of defense against external threats, determining pathogen 21.5 MJ/kg gross energy (Table 1). Both tested experimental adhesion to the epithelial surfaces (10, 11). Furthermore, diets were formulated to fulfill the nutritional requirements skin mucus participates in important physiological processes of juvenile gilthead seabream for summer conditions (18). like osmoregulation, swimming, sensory reception (12), and Diets were manufactured by Sparos Lda. (Olhão, Portugal). In ecological intra and interspecific interactions (11). Additionally, particular, main ingredients were ground (below 250 µm) in a the fish skin is also characterized by its active mucosal immunity, micropulverizer hammer mill (SH1; Hosokawa Micron, B.V., containing a skin-associated lymphoid tissue (SALT), which is Doetinchem, The Netherlands). Powder ingredients and oils able to respond in case of infection (12, 13). In fact, the immune were then mixed according to the target formulation in a paddle response described in fish skin against antigen stimulation is mixer (RM90; Mainca, S.L., Granollers, Spain). All diets were similar to other mucosa (14). That immune response involves manufactured by temperature-controlled extrusion (pellet sizes: the secretion of innate immune molecules and the action of 2.0 mm) by means of a low-shear extruder (P55; Italplast, S.r.l., specialized cells [(15) and references therein]. Parma, Italy). Upon extrusion, all feed batches were dried in a Therefore, the improvement of both the epidermal mucus convection oven (OP 750-UF; LTE Scientifics, Oldham, UK) for composition and the SALT response to environmental stressors 4 h at 45◦ C. including infective agents by means of dietary tools such as The nutritional assay was performed under natural functional feed additives, represents a promising approach for photoperiod (August–September), with daily monitoring of preventing bacterial-induced pathologies in farmed fish. In this the water temperature (25.1 ± 1.5◦ C, range: 22.6–28◦ C), oxygen context, the aim of the present study was to evaluate the (6.8 ± 1.7 mg/L; >80% saturation) (OXI330, Crison Instruments, inclusion of a functional feed additive composed by a blend of Barcelona, Spain) and pH (7.5 ± 0.01) (pHmeter 507, Crison Frontiers in Immunology | www.frontiersin.org 2 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity TABLE 1 | Formulation of the control diet used during the nutritional assay. selected from each tank (n = 12 fish per dietary treatment) and euthanized with an anesthetic overdose. A ca. 1 cm2 section of Ingredients Control diet (%) the skin from the mid region of the body over the lateral line of Fishmeal 70 LT FF Skagen 20.0 the right side from each fish was dissected, and the muscle tissue Fishmeal CORPESCA Super Prime 10.0 attached to it removed. Samples were immersed in RNAlaterTM CPSP 90 2.5 (Invitrogen, Thermo Fisher Scientific, Lithuania), incubated Squid meal 2.5 overnight (4◦ C) and stored at −80◦ C for further RNA extraction. Soy protein concentrate (Soycomil) 5.0 Wheat Gluten 5.0 Skin Transcriptomic Analysis Corn gluten 8.0 RNA Isolation and Quality Control Korfeed 60 4.5 Total RNA from the skin of twelve randomly selected fish Soybean meal 48 8.0 per dietary treatment was extracted using the RNeasy R Rapeseed meal 4.0 Mini Kit (Qiagen, Germany). Total RNA was eluted in a Sunflower meal 3.0 final volume of 35 µL nuclease-free water and treated with Wheat meal 7.0 DNAse (DNA-freeTM DNA Removal Kit; Invitrogen, Lithuania). Total RNA concentration and purity were measured using Whole peas 2.5 Nanodrop-2000 R spectrophotometer (Thermo Scientific, USA) Fish oil–COPPENS 9.0 and stored at −80◦ C until analysis. Prior to hybridization Soybean oil 1.5 with microarrays, RNA samples were diluted to 133.33 Rapeseed oil 2.5 ng/µL concentration, checked for RNA integrity (Agilent 2100 Vitamin and mineral Premix PV01 2.0 Bioanalyzer; Agilent Technologies, Spain) and selected by Soy lecithin–Powder 2.0 the criteria of a RIN value >8.5. Three different pools of Antioxidant powder (Paramega) 0.4 samples per dietary treatment were established (n = 4 fish Dicalcium phosphate 0.6 each pool). Proximate composition, % in dry basis Crude protein 46.2 Microarray Hybridization and Analysis Crude fat 18.4 Skin transcriptional analysis from both experimental groups Gross energy (MJ/kg) 21.5 was carried out using the Aquagenomics Sparus aurata oligonucleotide microarray v2.0 (4 × 44 K) (SAQ) platform. Detailed information and transcriptomic raw data are available at the Gene Expression Omnibus (GEO) public repository at Instruments). Salinity (35‰) (MASTER-20 T; ATAGO Co. Ltd, the US National Center for Biotechnology Information (NCBI), Tokyo, Japan), ammonia (0.13 ± 0.1 mg NH+ 4 /L), and nitrite accession numbers GPL13442, and GSE162504, respectively. (0.18 ± 0.1 mg NO− 2 /L) levels (HACH DR9000 Colorimeter, The sampling labeling, hybridization, washes, and scanning Hach R , Spain) were weekly measured. was performed as described in (19). Briefly, a one-color RNA labeling was used (Agilent One-Color RNA Spike-In Sampling kit; Agilent Technologies, USA). RNA from each sample pool At the end of the trial, all fish were anesthetized (buffered (200 ng) was reverse-transcribed with spike-in. Then, total 150 mg/L MS-222, Sigma-Aldrich, Madrid, Spain) and RNA was used as template for Cyanine-3 (Cy3) labeled cRNA measured for individual body weight and standard length synthesis and amplified with the Quick Amp Labeling kit (BWcontrol diet = 157.8 ± 14.2 g and SLcontrol diet = 17.3 ± 0.6 cm; (Agilent Technologies). cRNA samples were purified using the BWsupplemented diet = 150.8 ± 14.9 g and SLsupplemented diet = 17.1 RNeasy R micro kit (Qiagen). Dye incorporation and cRNA ± 0.6 cm) as published in (17). Then, eight fish from each tank yield were checked (NanoDrop ND-2000 R spectrophotometer). (n = 24 per dietary treatment) were randomly selected and skin Then, Cy3-labeled cRNA (1.5 mg) with specific activity >6.0 mucus sample collected following the method described in (19). pmol Cy3/mg cRNA was fragmented at 60◦ C for 30 min, In brief, skin mucus was collected from the over-lateral line of and hybridized with the array in presence of hybridization anesthetized fish in a front to caudal direction using sterile glass buffer (Gene expression hybridization kit, Agilent Technologies) slides, and mucus was carefully pushed and collected in a sterile at 65◦ C for 17 h. For washes, microarrays were incubated tube (2 mL), avoiding the contamination with blood and/or with Gene expression wash buffers, and stabilization and urine-genital and intestinal excretions. The above-mentioned drying solution according to manufacturer instructions (Agilent procedure lasted <2 min in order to avoid the degradation Technologies). Microarray slides were then scanned (Agilent of mucus metabolites. Mucus samples were homogenized G2505B Microarray Scanner System), and spot intensities using a sterile Teflon pestle to desegregate mucus mesh before and other quality control features extracted (Agilent Feature centrifugation at 14,000 × g during 15 min at 4◦ C. The resultant Extraction software version 10.4.0.0). mucus supernatants were collected, avoiding the surface lipid The Search Tool for the Retrieval of Interacting Genes layer, aliquoted, and stored at −80◦ C for further analysis. For (STRING) public repository version 11.0 (https://string-db. transcriptional analysis purposes, other four fish were randomly org) was used to generate the skin transcripteractome for the Frontiers in Immunology | www.frontiersin.org 3 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity fish fed the phytogenic-supplemented diet. A Protein-Protein at λ = 505 nm and lactate values expressed as µg lactate interaction (PPI) Networks Functional Enrichment Analysis for per mL of skin mucus. Protein concentration of previously all the differentially expressed genes (DEGs) was conducted homogenized mucus samples was determined using the with a high-confidence interaction score (0.9) using Homo Bradford assay (22) using bovine serum albumin (Sigma sapiens as model organism (17, 20). Gene Ontology (GO) and Aldrich, Madrid, Spain) as standard. In particular, mucus Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment samples or standard solutions (from 0 to 1.41 mg mL−1 ) analysis of all the DEGs obtained were also assessed through were mixed with 250 µL of Bradford reagent and incubated STRING (P < 0.05). In order to confirm match of gene acronyms for 5 min at room temperature. The OD was determined between both Homo sapiens and gilthead seabream species, at λ = 596 nm in a microplate reader. Protein values were human orthology identification based on gene/protein name expressed as mg protein per mL of skin mucus. Cortisol levels was accessed through the Genecards (www.genecards.org) (21) were measured using an ELISA kit (IBL International, Tecan and Uniprot (www.uniprot.org) databases. Additionally, protein- Group, Switzerland) following the manufacturer’s instructions protein BLAST (BLASTp) were run (E < 10−7 ; query cover for saliva determinations. Values of OD were determined at > 95%). λ = 450 nm with a microplate reader. Cortisol values were expressed as ng cortisol per mL of skin mucus. All standards Skin Mucus Parameters and samples were analyzed in triplicate (methodological Bacterial Growth Assessment in Skin Mucus replicates) and spectrophotometric measurements were Two bacterial fish pathogens were used for the growth conducted with an Infinity Pro200TM spectrophotometer curve assay, Vibrio anguillarum (CECT number: 522T), and (Tecan, Männedorf, Switzerland). Pseudomonas anguilliseptica (CECT number: 899T) from the Mucus ratios referred to protein (glucose/protein, Spanish Type Culture Collection (CECT, University of Valencia, lactate/protein and cortisol/protein) were calculated in order to Valencia, Spain), and the non-pathogenic bacterium for fish, avoid the putative dilution or concentration derived from mucus Escherichia coli (DSMZ number: 423) from the German sampling. As an indicator of the metabolic aerobic response, the Collection of Microorganisms and Cell Cultures (Leibniz glucose/lactate ratio was also calculated (19). Institute DSMZ, Germany). The two pathogenic bacteria were Ferric Antioxidant Power (FRAP) was measured by means cultured at 30◦ C for 24 h in marine broth (MB-2216, Becton of an enzymatic colorimetric test (Ferric antioxidant status and Dickinson, USA) and the E. coli was cultured at 37◦ C for detection kit, Invitrogen, Thermo Fisher Scientific, Spain), 24 h in trypticasein soy broth (TSB, Laboratorios Conda, Spain). following the manufacturer’s instructions for plasma, with For the culture in skin mucus, bacterial suspension with optical minor modifications. Briefly, 20 µL of mucus sample or density (OD) of 0.2 were centrifuged and the pellet resuspended standard solutions (from 0 to 1,000 µM µL−1 of FeCl2 ) were in sterile PBS, diluted in new growth medium and adjusted to 106 mixed with 75 µL of FRAP color solution and incubated colony-forming units (CFU) mL−1 . Then, to study the bacterial at room temperature for 30 min, in triplicate. The OD growth capacity in the skin mucus, aliquots of 100 µL of the was measured at λ = 560 nm. Antioxidant values were previously cultured bacteria were incubated in 100 µL of skin expressed as nmol FRAP per mL of mucus, and nmol mucus (3 pools of 6 individual fish per dietary treatment). In FRAP per mg of mucus protein. All measurements were parallel, 100 µL of the same cultured bacteria were also incubated performed with a microplate spectrophotometer reader (Infinity in 100 µL of its respective culture media, as a positive control. Pro200TM spectrophotometer). Triplicates of 100 µL of each fish mucus samples added to 100 µL of culture media were used as negative control and values subtracted from the bacteria–mucus aliquot results. The bacterial Statistical Analysis growth was measured by absorbance at λ = 400 nm every 30 min Differences between growth performance parameters were for 14 h at 25◦ C in flat-bottomed 96-well plates using an Infinity analyzed through an unpaired t-test (P < 0.05) with GraphPad Pro200TM spectrophotometer. Similar temperature values for PRISM 7.00 assuming data homoscedasticity. Differences bacterial growth cultures (25◦ C) and fish rearing (25.1◦ C) were between skin mucus metabolites and cortisol, and differences in chosen in order to standardize mucus analyses with regard to fish bacterial growth inhibition between the two dietary treatments rearing conditions used in the nutritional trial. were assessed with SPSS Statistics for Windows, Version 22.0 (IBM Corp, Armonk, NY, USA) through an unpaired t-test (P Skin Mucus Metabolites and Cortisol Analyses < 0.05). Microarrays extracted raw data were imported and Glucose concentration on fish skin mucus was determined by an analyzed with GeneSpring version 14.5 GX software (Agilent enzymatic colorimetric test (LO-POD glucose, SPINREACT R , Technologies). The 75% percentile normalization was used to St. Esteve de Bas, Spain) as described in (19). The OD of the standardize arrays for comparisons and data were filtered by reaction was determined at λ = 505 nm with a microplate expression. An unpaired t-test was conducted without correction reader and glucose values expressed as µg glucose per mL to identify those DEGs between both dietary treatments. A P < of skin mucus. Lactate concentration was determined by an 0.05 was considered statistically significant. The representation enzymatic colorimetric test (LO-POD lactate, SPINREACT R ) for the principal component analysis (PCA) and the hierarchical following the manufacturer’s instructions but with slight heatmap were generated using GeneSpring version 14.5 modifications for fish skin mucus (19). The OD was determined GX software. Frontiers in Immunology | www.frontiersin.org 4 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity RESULTS Skin Transcriptomic Profile Under present experimental conditions, in order to determine the modulatory effect of the dietary supplementation of a blend of garlic essential oil, carvacrol, and thymol upon the skin transcriptome, a microarray-based transcriptomic analysis was conducted in gilthead seabream. In total, 534 differentially- expressed genes (DEGs) were found in the skin from both experimental groups (P < 0.05; Supplementary Table 1). Among these, 393 genes were up-regulated with 390 belonging to the 1.0– 1.5-fold change (FC) interval. The other 3 DEGs were grouped in the 1.5 ≤ FC ≤ 2.0 interval. On the other hand, 141 genes were down-regulated (P < 0.05) and grouped in the range of−1.5 ≥ FC ≥ −1.0. Although genes were observed to be mostly up- regulated in the group fed with the blend of phytogenics (73.6% of DEGs), gene modulation was moderated in terms of fold- change intensity (Figure 1A). Common segregation among the pool samples within the same dietary treatment was observed in the hierarchical clustering for the skin transcriptomic response based in similitude patterns of the DEGs response (P < 0.05) (Figure 1B). The observed differential profile among dietary treatments is supported by the PCA analysis for the analyzed FIGURE 1 | Skin transcriptomic profile of gilthead seabream (Sparus aurata) samples (Figure 1C). fed a diet supplemented with a blend of garlic essential oil, carvacrol and When considering the complete list of annotated DEGs, thymol. (A) Differential expression analysis of the gilthead seabream skin a functional network (transcripteractome) containing 203 transcriptomic response fed a diet supplemented with a blend of garlic, nodes was generated (Figures 2, 3), which resulted in 341 carvacrol and thymol. (B) Hierarchical clustering for the control and phytogenic-supplemented diets, based in similitude patterns of the differentially interactions (edges). The remaining 331 DEGs, annotated expressed genes (DEGs) detected from three sample pools per dietary group. as unknown genes, were excluded from the analysis. The Data of the six microarrays are depicted, one for each represented pool. Both enrichment analysis identified in the transcripteractome increased and decreased gene expression pattern is shown in green and red, two main representative processes that were considered to respectively. All transcripts represented are statistically significant (P < 0.05). encompass the several Gene Ontology (GO) annotations (C) Principal component analysis (PCA) of the DEGs of gilthead seabream skin response to the control diet (yellow) and phytogenic-supplemented diet (pink). obtained (Supplementary Table 2), denoted as (a) Transcription Please refers to Supplementary Table 1 for details. Regulation, and (b) Secretory Pathway. The “RNA processing” biological process (GO:0006396; 12 up-regulated genes; 14 down-regulated genes) was the exclusive differentially regulated GO term for the skin of genes), and “proton-transporting two-sector ATPase complex” fish fed the diet supplemented with the additive (Figure 2). (GO:0016469; 1 up-regulated genes; 3 down-regulated genes). Molecular functions “protein binding” (GO:0005515) and In order to identify the pathways significantly impacted “protein-containing complex binding” (GO:0044877) were by the total DEGs obtained, the functional analysis of KEGG also obtained. pathways revealed also significant differences in the regulation In order to elucidate the location relative to the cellular of genes associated with “protein processing in endoplasmic structures in which the DEGs perform their function, reticulum” (hsa04141; 3 up-regulated genes; 5 down-regulated several cellular components were identified in the functional genes), “phagosome” pathway (hsa04145; 3 up-regulated genes; 5 network, representing the association between them (Figure 3). down-regulated genes), and “Vibrio cholerae infection” pathway The analysis included the, “ribonucleoprotein complex” (hsa05110, belonging to the “infectious disease: bacterial” group; (GO:1990904; 10 up-regulated genes; 9 down-regulated genes), 1 up-regulated gene; 4 down-regulated genes) in the skin of the “vesicle” (GO:0031982; 20 up-regulated genes; 20 down- group fed with the blend of tested phytogenics (Figure 4 and regulated genes), “transport vesicle membrane” (GO:0030658; Supplementary Table 3). 2 up-regulated genes; 5 down-regulated genes), “COPII-coated Additionally, from the total DEGs obtained from the skin ER to Golgi transport vesicle” (GO:0030134; 2 up-regulated transcriptomic profile of fish fed the phytogenic-supplemented genes; 3 down-regulated genes), “Golgi-associated vesicle” diet, a set of genes were selected by their involvement in (GO:000579810; 3 up-regulated genes; 4 down-regulated the “immune system process” (GO:0002376; 12 up-regulated genes), “endosome” (GO:0005768; 10 up-regulated genes; genes; 5 down-regulated genes). Among the processes related to 9 down-regulated genes), “vacuole” (GO:0005773; 9 up- immunity, “antigen processing and presentation of exogenous regulated genes; 12 down-regulated genes), “lysosome” peptide antigen via MHC class I” (GO:0002479; 1 up- (GO:0005764; 7 up-regulated genes; 7 down-regulated regulated genes; 2 down-regulated genes), “leukocyte activation” Frontiers in Immunology | www.frontiersin.org 5 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity FIGURE 2 | Functional biological network of the differentially expressed genes (DEGs) in the skin of juvenile gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of garlic essential oil, carvacrol, and thymol. Green-shaded nodes represent up-regulated genes and red-shaded nodes represent down-regulated genes. Graphic keys and network stats: number of nodes = 203; number of edges = 341; average node degree = 3.36; average local clustering coefficient = 0.4; expected number of edges = 286; PPI enrichment p-value = 0.000807. Gene Ontology (GO) definition, count of DEGs within the biological process and respective false discovery rate are described in the graphical figure legend (bottom). For details, please refers to Supplementary Table 2. (GO:0045321; 6 up-regulated genes; 1 down-regulated genes), response implies a functional protective mechanism against “regulation of NIK/NF-kappaB signaling” (GO:1901222; 2 up- pathogenic bacterial growth on skin mucus. Considering that our regulated genes; 1 down-regulated genes), “positive regulation data registered a specific response to Vibrio, we included in our of T cell cytokine production” (GO:0002726; 2 up-regulated analysis the fish pathogen V. anguillarum. In addition, we also genes; 0 down-regulated genes), and “regulation of T cell included as control P. anguilliseptica (another pathogenic marine proliferation” (GO:0042129; 2 up-regulated genes; 1 down- fish bacteria) and E. coli as non-pathogenic fish bacterium. regulated genes) biological processes were highlighted (Figure 5 When cultured with the skin mucus from fish fed the and Supplementary Table 4). phytogenic-supplemented diet, a reduction on the growth of the pathogenic bacteria V. anguillarum was observed (t-test; P Bacterial Growth Capacity on Skin Mucus < 0.05; Figures 6A,B). Growth decrease was recorded between The transcriptome response arose the modulation of genes 4 and 14 h of bacterial culture; the most accentuated decrease associated to immune processes involved in the response to in growth values compared with control diet (over 30%) infectious bacterial diseases. Thus, we evaluated whether such were found between 8 and 12 h (Figures 6A,B). Regarding Frontiers in Immunology | www.frontiersin.org 6 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity FIGURE 3 | Functional network representing cellular components related to secretory pathways of the differentially expressed genes (DEGs) in the skin of juvenile gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of garlic essential oil, carvacrol and thymol. Node colors indicate the cellular component for each DEG represented. Green-shaded nodes represent up-regulated genes and red-shaded nodes represent down-regulated genes. Graphic keys and network stats: number of nodes = 203; number of edges = 341; average node degree = 3.36; average local clustering coefficient = 0.4; expected number of edges = 286; PPI enrichment p-value = 0.000807. Gene Ontology (GO) definition, count of DEGs within the biological process and respective false discovery rate are described in the graphical figure legend (bottom). For details, please refers to Supplementary Table 2. P. anguilliseptica, a decline in bacterial growth was observed of culture (t-test; P < 0.05), with a maximum decrease in growth in both gilthead seabream skin mucus samples from fish fed of 50.2 ± 1.6% at 14 h (Figure 6D). the control and phytogenic-supplemented diets (Figures 6C,D). Gilthead seabream mucus from both nutritional groups However, P. anguilliseptica growth decline was observed to be showed a decrease of E. coli growth during all the culture more accentuated in the mucus from fish fed the phytogenic- period (Figures 6E,F), though the most emphasized bacterial supplemented diet than in that of the control group at 12–14 h growth decrease was observed in the initial interval of 4–8 h Frontiers in Immunology | www.frontiersin.org 7 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity FIGURE 4 | Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways networks of the differentially expressed genes (DEGs) in the skin of juvenile gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of garlic essential oil, carvacrol and thymol. (A) Gray cores represent the KEGG pathways significantly impacted by the DEGs obtained from the skin of fish fed the phytogenic-supplemented diet. Green-shaded nodes represent up-regulated genes and red-shaded nodes represent down-regulated genes. (B) Nodes colors indicate the KEGG pathways for each DEG represented within the overall transcripteractome. Pathways description, count of DEGs within each pathway and respective false discovery rate are described in the graphical figure legend. (C) Interactions exclusively among the DEGs within each KEGG pathway. Nodes colors indicate the KEGG pathways for each DEG represented. Pathways description, count of DEGs within each pathway and respective false discovery rate are described in the graphical figure legend. Green-shaded nodes represent up-regulated genes and red-shaded nodes represent down-regulated genes. For details, please refers to Supplementary Table 3. with decreased growth value of 60.3 ± 2.5%, reducing gradually The content of soluble protein was not significantly affected by (Figure 6F). the functional feed additive (t-test, P > 0.05). However, glucose, lactate and cortisol levels were observed to be significantly Mucus Stress Biomarkers lower in the mucus of gilthead seabream fed the phytogenic- The skin mucus stress-related biomarkers and their ratios, as supplemented diet (t-test, P < 0.05). No differences among well as the ferric antioxidant power are summarized in Table 2. dietary groups were observed in terms of the ferric skin mucus Frontiers in Immunology | www.frontiersin.org 8 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity FIGURE 5 | Functional biological network of differentially expressed genes (DEGs) recognized by their involvement in the immune response in the skin of juvenile gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of garlic essential oil, carvacrol, and thymol. Green-shaded nodes represent up-regulated genes and red-shaded nodes represent down-regulated genes. Graphic keys and network stats: number of nodes = 30; number of edges = 24; average node degree = 1.6; average local clustering coefficient = 0.4; expected number of edges = 9; PPI enrichment p-value = 1.25e-05. Gene Ontology (GO) definition, count of DEGs within the biological process and respective false discovery rate are described in the graphical figure legend (bottom left). For details, please refers to Supplementary Table 4. Gene description, respective acronym, fold-change intensity (FC), modulation (color scale), and P-value are described (right). antioxidant power (t-test, P > 0.05). When data on skin mucus metabolic, stress-related, and signal transduction proteins biomarkers were normalized with the protein content of the [(23–25); among others]. In addition to mucus continuous sample, only cortisol levels (cortisol/protein) were observed to be secretion and replacement (26), its components provide an significantly reduced in skin mucus from fish fed the phytogenic- impermeable capacity against most bacteria and other pathogens, supplemented diet when compared to the control diet (t-test, immobilizing them and inhibiting their proliferation before they P < 0.05). Metabolic aerobic response, measured in mucus as can contact epithelial surfaces (10). Importantly, an intimate glucose/lactate ratio, was neither significantly affected by the crosstalk between skin tissue and its exuded mucus in response dietary treatment. to stimulus has been recently proposed (27), reinforcing the coordinated response capacity that takes place in this mucosal- associated lymphoid tissue. DISCUSSION In the present study, we evaluated the protective benefits of a blend of dietary garlic essential oil, carvacrol, and thymol The fish skin mucosal surface is in direct contact with the aquatic on gilthead seabream in terms of skin transcriptome and skin environment and represents the first line of defense against mucus secretions. The potential increased skin mucus protective external threats, determining pathogen adhesion to the epithelial capacity observed in our gilthead seabream fed with phytogenic- surface (10, 12). As a mucosal tissue, the skin is characterized supplemented diet could be attributed to the exudation of a by its ability to produce mucus, which apart from being an variety of biologically active substances and several molecules intrinsic physical barrier, it contains glycosaminoglycans, lectins, of the innate and acquired immune system. In fact, many of antibacterial enzymes, immunoglobulins, and several structural, them have an already reported biostatic and biocidal activities Frontiers in Immunology | www.frontiersin.org 9 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity FIGURE 6 | Bacterial growth on skin mucus of juvenile gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of garlic essential oil, carvacrol and thymol, and a control diet devoid of the feed additive. Mucus samples were obtained from 3 pools of 6 individual fish. Data correspond to the mean ± SEM of triplicate bacterial growth curves of V. anguillarum (A), P. anguilliseptica (C) and E. coli (E). Gray circles correspond to bacteria growth in medium devoid of mucus; purple squares correspond to bacteria growth in the mucus of fish fed the control diet; and green rhombus correspond to bacteria growth in the mucus of fish fed the phytogenic-supplemented diet. Details on the statistical differences in bacterial growth between the experimental dietary groups is provided by the percentage of decrease in growth of V. anguillarum (B), P. anguilliseptica (D), and E. coli (F). Asterisks indicate significant differences in bacterial growth between dietary groups (t-test, P < 0.05). (28). Under this context, our transcriptomic analysis for the skin was observed to be up-regulated by the functional additive as revealed that several genes coding proteins potentially involved in well. Several calpain proteins were mapped in gilthead seabream’s an enhanced skin mucosal protective capacity. For instance, the epidermal mucus (34) while its presence in the skin mucus of H3 Histone Family Member 3A gene (h3f3a) was up-regulated cod (Gadus morhua) was suggested to be a key protective element in the skin of fish fed the phytogenic-supplemented diet. against V. anguillarum infection (35). Histones, full-length proteins and/or fragments, are recognized Iron is an essential micronutrient required for most bacteria antimicrobial molecules (29), which are found in the mucus to grow; in the host, this metal is associated to iron-binding of several fish species (30, 31), including gilthead seabream proteins, such as transferrin found in fish skin and mucus (23, (32, 33). Calpain 1 (capn1), a non-lysosomal cysteine protease, 30, 32), limiting its availability to invading pathogenic bacteria Frontiers in Immunology | www.frontiersin.org 10 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity TABLE 2 | Skin mucus stress biomarkers of gilthead seabream fed an inflammation and an increased protective capacity against lice experimental diet supplemented with a blend of garlic essential oil, carvacrol and infection (42). The hba1 and steap4 up-regulation in the skin thymol, and the control diet devoid of the feed additive (n = 24 per dietary observed in our study may be implicated in a reduction in treatment). iron availability in the skin surface and mucus of fish fed Diet the phytogenics, hampering pathogenic bacteria growth as observed in the potential mucus antibacterial capacity observed Control Phytogenics in our study. Skin mucus stress biomarkers Fish SALT is characterized by both humoral and cellular Protein (mg/mL) 16.73 ± 2.22 12.86 ± 1.77 components which intimately communicate to mount an Glucose (µg/mL) 13.43 ± 3.95 6.88 ± 1.63* immune response in which both innate and adaptive defense Lactate (µg/mL) 9.80 ± 2.62 3.62 ± 1.09* mechanisms are involved (15). For instance, the Interferon Cortisol (ng/mL) 3.47 ± 0.85 0.35 ± 0.05* Related Developmental Regulator 1 (ifrd1) was up-regulated FRAP (µmol/mL) 1,923 ± 244 1,790 ± 315 by the functional diet. Ifrd1 gene encodes a protein related to Skin mucus stress biomarkers ratios interferon family. In fish is widely recognized by its involvement Glucose/Protein (µg/mg) 0.74 ± 0.16 0.60 ± 0.17 in the innate immune antiviral response (43), while it is Lactate/Protein (µg/mg) 0.46 ± 0.05 0.31 ± 0.12 also highly expressed in differentiating neutrophils, playing an Glucose/Lactate (mg/mg) 0.69 ± 0.14 0.77 ± 0.15 important role in neutrophils effector function (44). An increase Cortisol/Protein (ng/g) 208 ± 48.80 31.85 ± 7.03** of ifrd1 transcripts was observed in the skin of zebrafish (Danio FRAP/Protein (µmol/mg) 114 ± 16.9 139 ± 23.1 rerio) infected by Aeromonas hydrophila (45), whereas its up- regulation was also related to its role in the immediate response Asterisks indicate significant differences between experimental diets (*P < 0.05, **P < of the fish immune system to stress (46). Moreover, transcripts of 0.01; t-test). the Complement Factor H (cfh) increased in the skin of gilthead seabream fed the phytogenic-supplemented diet. CFH, a major (36). For instance, V. anguillarum iron-uptake system is crucial regulator of the complement system, is essential for directing for sequestering iron from these proteins and accomplishing the complement system toward pathogen-related infections, skin colonization and penetration (37). Interestingly, some since its transcription is induced by lipopolysaccharide (LPS) genes related with iron metabolism were observed to be down- (47), whereas it increases the contact between neutrophils regulated in the skin of fish fed the functional diet. In our and pathogens, increasing cell’s phagocytosis capacity and transcriptional analysis, the Iron-Responsive Element-Binding antimicrobial activity (48). CFH is also reputed for protecting Protein 2 (ireb2) gene was down-regulated in fish fed the host cells and tissues from the self-innate immunity (49). This phytogenic-supplemented diet. When cell’s iron levels are low occurs due to the interaction of the factor H with the C3 or depleted, this RNA-binding protein binds to iron-responsive convertase and the C3b component (50). Although CFH is elements, found for instance in transferrin mRNAs, regulating predominantly expressed in the liver compared to other tissues the translation and stability of those mRNAs and consequently and organs like the muscle, intestine, fins, eyes, and gills (47), regulating iron availability (38). In zebrafish, the increase of present data indicate that the skin may also play a relevant role ireb2 expression in spleen was proposed to be linked to an in the regulation of the alternative pathway of complement in augmented iron uptake from V. anguillarum (39). Hence, the skin secretions. This data is in agreement with different studies down-regulation of ireb2 observed in the skin of fish fed that have identified several complement factors in fish skin the phytogenic-diet could be suggesting a decrease in the mucus secretions for several species (30, 51), including gilthead cellular iron uptake as a consequence of the reduction of seabream (23, 24). pathogenic bacteria load in the mucosal tissue. This hypothesis Our study also revealed that Cathepsin A (ctsa) was up- is supported by the decreased growth capacity for pathogenic regulated in the skin of fish fed the phytogenic-supplemented bacteria in skin mucus observed in the fish fed the tested diet. An increase in turbot (Scophthalmus maximus) ctsa functional diet. expression in skin was described in response to infection On the other hand, iron plays an important role in challenges, while ctsa genes microbial binding capacity was also hemoglobin production. The Hemoglobin Subunit Alpha (hba1) observed, in which a high affinity to LPS, and a lower affinity to was up-regulated in the skin of fish fed the phytogenic- lipoteichoic acid (LTA) and peptidoglycan (PGN), was suggested supplemented diet. Hemoglobin functionality is not restricted to be implicated in the sensing and phagocytosis of bacterial to oxygen transport, since it also binds to pathogen-associated pathogens (52). By contrast, in our study the expression of molecular patterns (PAMPs), triggering an immune Toll-like Cathepsin L (ctsl) was down-regulated. Different cathepsins have receptor (TLR)-mediated signal transduction (40). In addition, been detected in the mucus of several fish species, which were the STEAP4 Metalloreductase (steap4) transcripts increased observed to exhibit high bacteriolytic activity against several fish by the phytogenic-diet. This metalloreductase is involved in pathogens (53, 54), evidencing their key role in fish mucosal iron and copper homeostasis, playing also a role in the innate immunity. Fish skin ctsl transcripts were observed to be protection against inflammatory-mediated cellular damage (41). significantly up-regulated after challenges with several bacterial In Atlantic salmon (Salmo salar) skin, steap4 was up-regulated by pathogens, including V. anguillarum (54, 55). Moreover, as for dietary phytogenics, which was associated to iron sequestration, ctsa genes, it was demonstrated that ctsl genes have strong Frontiers in Immunology | www.frontiersin.org 11 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity in vitro binding capacity to microbial ligands, suggesting an transport to degradative compartments (65) and (iii) in important role of ctsl in fish mucosal immunity (55). The the control of membrane trafficking along the phagocytic different gene expression pattern observed for ctsa and ctsl could pathway (66). In addition, the Microtubule Associated Protein be related to a time-dependent response, as suggested previously 1 Light Chain 3 Gamma (map1lc3c) was up-regulated. This for genes involved in the immune response in fish subjected autophagy-related protein is involved in the LC3-associated to feeding trials (56). The differential expression between both phagocytosis, in which LC3 is recruited to the phagosome cathepsins could be also attributed to the mucosal tissue response membrane during phagocytosis of pathogens, enhancing the specificity. In this way, it has been reported that the same fusion between phagosome and lysosomes (67). In fact, fish stimulus may differentially modulate the expression for the same epidermal macrophages are characterized by well-developed genes depending of the mucosal tissue evaluated (57). However, endoplasmic reticulum and Golgi areas and several lysosome-like if both genes are linked with the decrease in the bacterial growth vesicles and phagosomes (68). Remarkably, the up-regulation of observed at skin mucus deserves further investigations. the MAF BZIP Transcription Factor B (mafb), a myeloid lineage- Fish professional phagocytes include macrophages, specific transcription factor, which expression levels increase granulocytes, dendritic cells and B cells, and as for other during macrophage differentiation and maturation (69), was also vertebrates, phagocytosis in fish is recognized as a critical observed in the skin of fish fed the phytogenic-supplemented component of the innate and adaptive immune responses against diet. Therefore, the regulation of LC3 proteins-coding genes and pathogens, known to elicit several antimicrobial mechanisms. the up-regulation of mafb by the phytogenics might support the Under this context, the KEGG “Phagosome” pathway obtained participation of phagocytic cells in the immune response from the from our functional analysis suggests the modulation of skin observed in our study. The down-regulation of Microtubule phagocytic events by the administered phytogenics. For instance, Associated Protein 1 Light Chain 3 Beta (map1lc3b) opens the despite the extracellular roles of cathepsins (58), these proteins possibility to selective and differential mechanisms of activation are mainly found in endolysosomal structures where they are aimed to the promotion of the phagocytic activity in response to crucial for protein degradation and Major Histocompatibility phytogenic supplementation. Complex (MHC) Class II mediated immune responses (59). Garlic, carvacrol, and/or thymol have been several times Interestingly, the MHC Class II Alpha gene (mhc-IIa) was described to improve immune cells phagocytic capacity. For the second most up-regulated gene in the skin of fish fed instance, dietary garlic (0.5 and 1% inclusion) enhanced the the phytogenic-supplemented diet. While MHC-IIa protein activity of head kidney macrophages phagocytic in rainbow trout was identified in gilthead seabream skin mucus proteome (Oncorhynchus mykiss) (70). Similar results were observed for (34), the main function of fish MHC Class II molecules blood leukocytes of juvenile hybrid tilapia (Oreochromis niloticus is to present the peptides generated in the endolysosomal x Oreochromis aureus) fed a 0.5% garlic-supplemented diet structure on the cell surface of B cells and phagocytes for (71). Likewise, carvacrol and thymol supplementation (0.2%) in their recognition by the CD4+ T cells (60). In fact, the juvenile hybrid tilapia’s diet significantly enhanced phagocytosis gilthead seabream acidophilic granulocytes, considered the of head kidney macrophages (72). The phagocytic activity of main professional phagocytic cell type for this fish species, serum leukocytes from common carp (Cyprinus carpio) was were demonstrated to show high mhc-IIa gene expression increased due to dietary oregano’s essential oil, which is rich (61). Moreover, they have also proved to have phagocytic in carvacrol and thymol, in a dose-dependent manner (73). A activity against bacterial pathogens such V. anguillarum similar enhanced head kidney leukocytes’ phagocytosis was also (62), being able to release antimicrobial peptides into the found in gilthead seabream fed a diet supplemented with oregano phagosome of the ingested pathogenic bacteria (63). The powder (0.5 and 1%) (74). “leukocyte activation”, “regulation of T cell proliferation”, Phagocytic events are driven by rearrangements of the actin and “antigen processing and presentation of exogenous cytoskeleton (75). Calponin 2 (cnn2), an actin cytoskeleton- peptide antigen via MHC class I” biological processes obtained associated regulatory protein that restricts the pro-inflammatory from our enrichment analysis, might suggest the activity of activation of macrophages (76), was up-regulated in the skin acidophilic granulocytes and/or other immune cells in the skin by the phytogenics. Similarly, the Actin Beta (actb) gene was of gilthead seabream fed the functional diet. Similarly, previous also up-regulated. Actin is commonly found in the mucus of transcriptional results on the effect of the same functional several fish species, including gilthead seabream (23, 32), which feed additive in gilthead seabream mucosal tissues such gills has led to speculations on its immune function in fish defense. (17) suggested the recruitment and activation of acidophilic In fact, ACTB levels were observed to be significantly increased granulocytes as a consequence of the immunostimulatory effect in sea lice challenged Atlantic salmon (30, 51). In gilthead of this additive. seabream, actb expression in skin mucus was favored by a dietary The functionality and modulation of genes related to the probiotic administration (24). Furthermore, the extracellular “endosome” and “lysosome” cellular components reinforce the cytoplasmic actin in insects was observed to bind to bacteria hypothesis of an increased professional phagocytic activity surface, mediating its phagocytosis and killing (77), suggesting in the skin of fish fed the phytogenic-supplemented diet. that actin could be functionally active in fish skin mucus Among them, Rab-interacting proteins coding genes (rilp, as well. However, this hypothesis needs further investigation. rab11fip1) were up-regulated. These genes are involved in In summary, our transcriptional analysis could be indicating several processes like (i) endosomal recycling (64), (ii) endocytic an enhanced phagocyte function in the skin of fish fed the Frontiers in Immunology | www.frontiersin.org 12 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity phytogenic-diet, which would suggest the promotion of the host’s (89), demonstrating its important role in the fish innate defense ability in resisting bacterial infections. immune response and homeostasis maintenance. The DEAD- In order to prevent the stable colonization of potential Box Helicase 1 (ddx1), reported to enhance NF-kB mediated pathogens, mucus is continuously synthesized, secreted and transcriptional activation (90) and recently associated to antiviral replaced (26). This continuous regulation of mucus secretions responses in fish (91), was also down-regulated in the skin of represents one of the first barriers against potential pathogens fish fed the phytogenic-supplemented diet. Therefore, according and toxins (10). Accordingly, our functional analysis determined to the overall response observed considering the transcriptomic the modulation of the KEGG “Protein processing in endoplasmic profiling of il-1β, aimp1, tnfaip2, tnip2, nrros, nlrc3, and ddx1, the reticulum” pathway and several cellular components connected regulation of these pro- and anti-inflammatory genes suggests an to the secretory pathway, such as transport vesicles, Golgi- active cytokine secretion, an immune cell-cell signaling, and the associated vesicles and vacuoles. The regulation of the secretory tight control of such response. machinery could be supporting the active biosynthesis and Regardless of its critical function protecting the host, the release of immune-related factors on the skin mucus that would skin mucus also represents an important portal of entry for mediate in the response observed in our study. The mucus pathogens since it can provide a favorable microenvironment secretion is a complex process that represents the endpoint of for some bacteria, the main disease agents for fish, which the interaction between the innate immune system, endocytosis may induce the development of biofilms depending on the and autophagy events, ROS generation and mucin secretion pathogen adhesion capacity (10). Interestingly, the down- (78). From the immunological point of view, cytokines, and regulation of genes of the KEGG “Vibrio cholerae infection” chemokines are molecules trafficked in secretory granules and pathway was obtained from the analysis of the DEGs of fish vesicles through secretory pathways in immune cells (79) fed the phytogenic-based additive. Although the analysis was that could be also involved in the response observed. Under performed using Homo sapiens as model organism, the down- this context, transcripts of the pro-inflammatory cytokine regulation of several genes of the “Vibrio cholerae infection” Interleukin 1 Beta (il-1β) were observed to be increased in KEGG pathway could also be applied, from a comparative point the skin of fish fed the phytogenic-supplemented diet. In of view, to an infection process involving a Vibrio species in fish, IL-1β is a recognized chemoattractant for leukocytes a mucosal tissue, such as V. anguillarum, as a consequence of (80). Additionally, the Aminoacyl tRNA Synthetase Complex the protective effect of the additive. For instance, the down- Interacting Multifunctional Protein 1 (aimp1) gene was also regulation of several coding genes for V-type H+ ATPase up-regulated. Secreted AIMP1 possesses inflammatory cytokine subunits (atp6v1c1, atp6v1b1, atp6ap1) in fish fed the phytogenic- activity responsible for activating monocytes and inducing the supplemented diet was obtained. The V-type H+ ATPase production of pro-inflammatory cytokines, mainly the Tumor complex in endosomes and lysosomes is responsible for the Necrosis Factor (TNF) (81). In accordance, genes that are vesicle import of protons and maintenance of the internal acidic activated in response to the pro-inflammatory cytokine TNF, pH, crucial for degradative enzymes activity (92). Apart from such the TNF Alpha Induced Protein 2 (tnfaip2) and TNFAIP3- the “proton-transporting two-sector ATPase complex” cellular Interacting Protein 2 (tnip2) were also up-regulated by the component obtained from our enrichment analysis, the down- phytogenics in our study. The last inhibits the NF-kB pathway regulation of the above-mentioned genes also figured in the activation (82), negatively regulating the transcription of other KEGG “Phagosome” pathway, which could be suggesting a pro-inflammatory cytokines and, consequently, controlling the decreased phagosomal activity at the sampling time evaluated inflammatory response. Thus, such response could be intimately (65 days of diet administration). Such gene modulation could related with the role of AIMP1 in dermal fibroblast proliferation be associated to a decrease in skin bacterial pathogens in our and wound repair (83). fish fed with phytogenic-supplemented diet. This hypothesis is Other mediators of the inflammatory response were also in agreement with the results obtained from the bacterial growth observed to be regulated in the skin of fish fed the phytogenic- assessment in the epidermal mucus in vitro. diet. For instance, the Negative Regulator of Reactive Oxygen Bacterial growth in gilthead seabream mucus was evaluated Species (nrros) gene expression was increased. The NRROS for both dietary groups, control and phytogenic-supplemented protein regulates ROS production by phagocytes during diets, using two pathogenic fish bacteria, V. anguillarum and P. inflammatory response, allowing phagocytes to produce high anguilliseptica, as well as a non-pathogenic fish bacterium, E. coli. amounts of ROS in case of infection, while minimizing host’s Our study revealed that the tested feed additive reduced bacteria tissue damage (84). NRROS is also suggested to play a role in the growth capacity, suggesting an enhanced skin mucus inhibitory maintenance of the immune homeostasis through the inhibition capacity against both V. anguillarum and P. anguilliseptica. The of TLR-mediated MAPK and NF-kB activation (85). Another chosen pathogenic bacteria are widely recognized as disease negative mediator of the inflammatory response, the NLR Family causing agents in several fish species (93, 94), including gilthead CARD Domain Containing 3 (nlrc3) was also observed to be up- seabream (95). The observed reduced growth capacity of V. regulated in fish fed the phytogenic-supplemented diet. NLRC3 anguillarum in the skin mucus of gilthead seabream fed the is known to negatively regulate NLR-mediated inflammatory phytogenic-supplemented diet is especially relevant because of responses (86). In several fish species, the overexpression of the evidence that Vibrio strains exhibit a chemotactic response nlrc3 was observed to be systematically induced by bacterial to mucus (37). In the last years, disease records indicate and LPS challenges (87, 88), including in mucosal tissues that Vibrio spp. infections are the most common bacterial Frontiers in Immunology | www.frontiersin.org 13 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity infections in gilthead seabream, mostly reported during the most Gram positive and negative bacteria, including E. coli (101). hatchery phase (96). Besides, P. anguilliseptica is one of the Nevertheless, the absence of an inhibitory response against E. coli main agents responsible for outbreaks associated with “winter growth in our study could be suggesting the promotion of the disease” in gilthead seabream farming, being considered a more skin innate immunity against fish pathogens (102). opportunistic pathogen whose infections in gilthead seabream From a physiological perspective, bacterial pathogens such usually occur when fish are under environmental stress (97). V. anguillarum are able to elicit strong cortisol-mediated stress The different growth curves behavior of V. anguillarum and P. responses when they adhere to the mucosal surface (36). For anguilliseptica in gilthead seabream mucus may be attributed this reason, in this current study we also measured classic skin to different virulence of pathogenic bacteria, differences in mucus stress biomarkers in order to establish a correlation chemotaxis to skin mucus and adherence capacity. between the gene expression profile, antibacterial response Our results suggest that the phytogenic-supplemented diet and the fish physiological status. Therefore, only cortisol was may reduce the settlement of the studied bacteria in the skin observed to be significantly reduced in fish fed the phytogenic- surface, decreasing the risk of infection. Previous nutritional supplemented diet. Although the exact mechanisms involved in studies in which diets containing garlic, carvacrol, or thymol cortisol exudation through fish mucus are still unclear, cortisol provided effective mucus antibacterial characteristics against is the main glucocorticoid and the final product of the HPI fish pathogens. For instance, dietary garlic supplementation (5 axis response to stress, varying considerably among species and and 10 g kg-1) was demonstrated to significantly increase skin according to the duration and severity of the stressor (103). mucus antimicrobial activity against several bacterial pathogens Cortisol decrease also favors the fish local mucosal immunity, in the freshwater Caspian roach (Rutilus rutilus) (98). A similar promoting more effective defense responses against pathogens increased bactericidal activity against Photodamselae subsp. (104). Besides the hypothesized decrease in skin pathogenic piscicida was observed in the skin mucus of gilthead seabream bacteria and its potential impact on cortisol-induced responses, juveniles fed diets supplemented with oregano powder (0.5 and and vice versa, several phytogenic active substances have been 1% inclusion) for 15 and 30 days (74). In addition, an enhanced reported to have sedative properties in fish (105). For instance, skin mucus bactericidal activity against V. parahaemolyticus garlic powder inclusion in common carp diet (0.5, 1, and and Aeromonas hydrophila was observed in Pacific red snapper 1.5%) decreased plasma cortisol and glucose levels, mitigating (Lutjanus peru) fed a diet supplemented with a medicinal ammonia stress-induced effects (106). Similar results were plant extract rich in carvacrol and thymol (0.5, 1, and 2% obtained in rainbow trout fed 3% garlic powder supplemented inclusion) (99). On the contrary, other studies incorporating diets (107). Accordingly, the dietary supplementation of a Oliveria decumbens, which is rich in carvacrol (18.8 and 52.9% similar additive containing garlic and Labiatae plant essential included in an essential oil and aromatic water fractions, oils (0.02% inclusion) reduced significantly plasma cortisol respectively) and thymol (20.3 and 37.6% included in an levels in European seabass (Dicentrarchus labrax) challenged essential oil and an aromatic water fractions, respectively), in by confinement (108). Although the diet effect on fish stress Nile tilapia (O. niloticus) diets reported the absence of changes response is usually evaluated in blood, a positive correlation in mucus bactericidal activity against Streptococcus iniae. By between cortisol levels on plasma and fish mucus was contrast, the compounds showed high antibacterial capacity demonstrated (109, 110) including for gilthead seabream (111). when evaluated in vitro (100). Similarly, Beltrán et al. (74) Therefore, the observed decrease in mucus cortisol may suggest did not find an enhancement of antibacterial activity against a decrease in the allostatic load due to the properties of the V. anguillarum in the mucus of gilthead seabream fed a diet phytogenics used in this current study and/or as a consequence supplemented with Origanum vulgare at 0.5 and 1.0%, although a of the promotion of the non-specific innate immunity, although significant increased bactericidal activity against Photobacterium these two hypotheses are not mutually excluding. damselae subsp. piscicida was observed. The above-mentioned During stress adaptation, cortisol has been suggested as a contradictory results could be a consequence of different factors, signal factor that induces tissue specific molecular programming such as different phytogenics and their content in bioactive in fish (112). Cortisol is able to induce a skin local stress compounds, supplementation period of the functional diet, response in fish (57, 94, 109, 113), which is particularly dietary compounds delivery and bioavailability, or an evidence of characterized by the increase of the secretory activity, related the synergy between the phytogenics tested that might improve vesicles, apoptosis (114), and transcriptional alterations (57). such bactericidal capacity. Under this context, the observed changes in mucus cortisol Moreover, the E. coli, a non-pathogenic bacterium for fish, was secretion might be contributing to the obtained secretory-related also used as an indicator of the potential antibacterial capacity of transcriptional response as well. Furthermore, since a correlation the skin mucus, neglecting a potentially acquired immunization. between cortisol secretion and skin mucus oxidative stress was Although no significant differences were observed among the demonstrated (115), a reduction of the skin oxidative stress in experimental diets, a decreasing trend in E. coli growth was response of an increase in antioxidative power induced by the observed due to the presence of mucus from both dietary groups tested phytogenics would be also expected. In fact, in our current in the culture medium. Interestingly, garlic-supplemented diets study genes coding for antioxidative enzymes were observed caused a significant increase in the Caspian roach fry skin mucus to be up-regulated in the skin of fish fed the phytogenic- antibacterial activity against E. coli when compared to the control supplemented diet, as for example the Glutathione S-Transferase group (98). In addition, carvacrol and thymol were reported to Kappa 1 (gstk1). GSTK1 belongs to the as Glutathione S- have a bacteriostatic and bacteriolytic in vitro activity against Transferase (GST) superfamily of oxidative stress enzymes, Frontiers in Immunology | www.frontiersin.org 14 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity which are mainly known for their important role in cellular diet induces the activation of the mucosal immune response that detoxification (116). GSTs have been used as markers for promotes the secretion of non-specific immune molecules into fish antioxidative capacity (117), including the evaluation of the skin mucus, resulting in the decrease of bacterial growth phytogenics in aquafeeds (118). Under stress-imposed conditions capacity in mucus. From our transcriptomic enrichment analysis, or injury, the transcription of skin gst is usually decreased and the regulation of genes related with the secretory pathway associated to immunosuppression in gilthead seabream (33). suggests that the tested phytogenics could be also stimulating The Nuclear Factor Erythroid 2 Like 2 (nfe2l2), an important the recruitment of phagocytic cells. The reduction in skin transcription factor that positively regulates the expression of mucus cortisol is in line with the recognized properties of the cytoprotective genes (119), and the N-Acetyltransferase 1 (nat1), phytogenics. Since the exact mechanisms promoted by the tested known for its participation in the detoxification of drugs and phytogenics were not yet demystified, further analysis should other xenobiotics (120), were also observed to be up-regulated be made in order to assess the effect of the experimental diet by the functional diet. In addition, Heat Shock Protein family on skin phagocytes and phagocytosis potential. More efforts genes (dnajc8, dnajb9, and hspa14) were down-regulated in the are also needed for determining the impact of the functional fish fed the phytogenic-supplemented diet. In particular, HSPA14 additive on the skin defensive status against other pathogenic is member of the Hsp70 family, which proteins levels have bacteria that threaten the success of aquaculture under intensive been described to increase in fish under stress or pathological farming regime. conditions (121, 122). Therefore, the regulation of these genes is supporting the involvement of immune cells in the skin response DATA AVAILABILITY STATEMENT observed, suggesting a reduction of the skin oxidative stress in response of the reduced mucus cortisol secretion and/or by the The datasets presented in this study can be found in online antioxidative characteristics of the tested phytogenics. In fact, the repositories. The names of the repository/repositories inclusion of garlic (106, 123), carvacrol, and thymol (73, 124) and accession number(s) can be found in the in aquafeeds have been continuously demonstrated to enhanced article/Supplementary Material. fish antioxidant status. In the present study, the epidermal mucus antioxidant capacity was also measured by mean of the FRAP analysis (102, 115). Although our transcriptional analysis ETHICS STATEMENT revealed the regulation of several markers of oxidative stress, IRTA facilities, where the trial was conducted, are certified and according to FRAP’s analyses the mucus antioxidant power was have the necessary authorization for the breeding and husbandry not significantly changed by the tested additive. Our results are of animals for scientific purposes. All procedures involving the in agreement with some previous studies which reported that handling and treatment of the fish were approved as far as the the dietary supplementation of carvacrol and/or thymol-rich care and use of experimental animals are concerned, by the compounds did not affect the skin mucus biochemical contents European Union (86/609/EU), the Spanish Government (RD or mucus antioxidant status (74, 100). 1201/2005), and the University of Barcelona (Spain). Collectively, these data clearly suggest a relationship between the tested phytogenics, the increased skin innate immunity and a cortisol-mediated response, promoting the overall animal AUTHOR CONTRIBUTIONS welfare. In fact, according to the only biological process significantly regulated among the experimental diets, the RNA Biological samplings were performed by EG, JF, RS, LF-A, IS, processing biological process, several genes implicated in and AI. The transcriptomic data analysis and interpretation was ribosomal proteins synthesis (riok1, rcl1, rrp1, nop56, nsa2, performed by JF, RS, EV-V, and FER-L. LF-A, IS, and AI carried tsr2) were mainly up-regulated in fish fed the phytogenic- out the skin mucus analyses. The study was supervised by EG, supplemented diet. Since ribosome biogenesis is the cell’s FER-L, and LT. JF wrote the original draft. Funding was obtained most costly process in terms of energy expenditure, this by EG and LT. All the authors provided critical feedback, read, process must be tightly regulated in order to avoid wasted and agreed to the published version of the manuscript. energy (125). Consequently, the up-regulation of such genes could be suggesting less stressed cells able to direct their FUNDING energy into this process. Since skin from the control group appear to be more susceptible to be colonized by pathogenic This work has been supported by the project Nutritional bacterial strains, it could be spending more energy in defense strategies for the improvement of productive performance: mechanisms and bacterial clearance than fish fed the phytogenic- the use of functional feeds and health diets in aquaculture supplemented diet. (DIETAplus), funded by JACUMAR (Ministry of Agriculture, In summary, our analysis of the skin transcriptional profiling Fisheries and Environment of Spain, MAPAMA) and FEMP as well as the skin mucus biomarkers and lower pathogenic (EU). Collaboration between Ibero-American researchers has bacterial growth capacity revealed a multifactorial response to been done under the framework of the LARVAplus network the dietary administration of garlic essential oil, carvacrol, and Strategies for the development and improvement of fish larvae thymol, mainly through the transcriptional regulation of factors production in Ibero-America (117RT0521) funded by the Ibero- of the innate immunity and the stimulation of the secretory American Program of Science and Technology for Development pathway. Our results suggest that the phytogenic-supplemented (CYTED, Spain). The funder bodies were not involved in Frontiers in Immunology | www.frontiersin.org 15 March 2021 | Volume 12 | Article 633621 Firmino et al. Phytogenics Promote Skin Innate Immunity the study design, collection, analysis, interpretation of data, ACKNOWLEDGMENTS the writing of this article or the decision to submit it for publication. FER-L thanks the support of Fondecyt regular We thank Dr. Alicia Estevez who led and supervised the grant (Nb. 1211841; ANID; Goverment of Chile). JF have nutritional assay. been subsidized by the Industrial PhD program of Generalitat de Catalunya and TECNOVIT-FARMFAES S.L. (Nb. 2017 SUPPLEMENTARY MATERIAL DI 017). EV-V was financially granted with DICYT-USACH Postdoctoral fellowship (Nb. 022043IB). Héctor R. Salomón The Supplementary Material for this article can be found Figueredo is supported by a Ph.D. grant from the government of online at: https://www.frontiersin.org/articles/10.3389/fimmu. Paraguay (BECAL). 2021.633621/full#supplementary-material REFERENCES 17. Firmino JP, Vallejos-Vidal E, Sarasquete C, Ortiz-Delgado JB, Balasch JC, Tort L, et al. Unveiling the effect of dietary essential oils supplementation 1. Assefa A, Abunna F. 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Conflict of Interest: JF is a current TECNOVIT-FARMFAES S.L employee doi: 10.1016/j.aquaculture.2017.10.031 conducting an Industrial Ph.D. 116. Li J, Xia Z, Ding J. Thioredoxin-like domain of human κ class glutathione transferase reveals sequence homology and structure similarity to the The remaining authors declare that the research was conducted in the absence of θ class enzyme. Protein Sci. (2005) 14:2361–9. doi: 10.1110/ps.0514 any commercial or financial relationships that could be construed as a potential 63905 conflict of interest. 117. Vallejos-Vidal E, Reyes-Cerpa S, Rivas-Pardo JA, Maisey K, Yáñez JM, Valenzuela H, et al. Single-nucleotide polymorphisms (SNP) mining and Copyright © 2021 Firmino, Fernández-Alacid, Vallejos-Vidal, Salomón, Sanahuja, their effect on the tridimensional protein structure prediction in a set Tort, Ibarz, Reyes-López and Gisbert. This is an open-access article distributed of immunity-related expressed sequence tags (EST) in Atlantic salmon under the terms of the Creative Commons Attribution License (CC BY). The (Salmo salar). Front Genet. (2020) 10:1406. doi: 10.3389/fgene.2019. use, distribution or reproduction in other forums is permitted, provided the 01406 original author(s) and the copyright owner(s) are credited and that the original 118. Hoseinifar SH, Shakouri M, Yousefi S, Van Doan H, Shafiei S, Yousefi publication in this journal is cited, in accordance with accepted academic practice. M, et al. Humoral and skin mucosal immune parameters, intestinal No use, distribution or reproduction is permitted which does not comply with these immune related genes expression and antioxidant defense in rainbow trout terms. Frontiers in Immunology | www.frontiersin.org 19 March 2021 | Volume 12 | Article 633621