REVIEW published: 18 June 2021 doi: 10.3389/fimmu.2021.695973 Phytogenic Bioactive Compounds Shape Fish Mucosal Immunity Joana P. Firmino 1,2,3, Jorge Galindo-Villegas 4*, Felipe E. Reyes-López 5,6,7 and Enric Gisbert 1 1 Aquaculture Program, Institut de Recerca i Tecnologia Agroalimentàries (IRTA) Centre de Sant Carles de la Ràpita (IRTA- SCR), Sant Carles de la Ràpita, Spain, 2 PhD Program in Aquaculture, Universitat Autònoma de Barcelona, Bellaterra, Spain, 3 R&D Technical Department, TECNOVIT – FARMFAES, S.L., Alforja, Spain, 4 Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway, 5 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain, 6 Facultad de Medicina Veterinaria y Agronomı´a, Universidad de Las Américas, Santiago, Chile, 7 Consorcio Tecnológico de Sanidad Acuı´cola, Ictio Biotechnologies S.A., Santiago, Chile Aquaculture growth will unavoidably involve the implementation of innovative and Edited by: sustainable production strategies, being functional feeds among the most promising Javier Santander, ones. A wide spectrum of phytogenics, particularly those containing terpenes and Memorial University of Newfoundland, organosulfur compounds, are increasingly studied in aquafeeds, due to their growth Canada promoting, antimicrobial, immunostimulant, antioxidant, anti-inflammatory and sedative Reviewed by: Seyed Hossein Hoseinifar, properties. This trend relies on the importance of the mucosal barrier in the fish defense. Gorgan University of Agricultural Establishing the phytogenics’ mode of action in mucosal tissues is of importance for Sciences and Natural Resources, Iran Débora Torrealba, further use and safe administration. Although the impact of phytogenics upon fish Pontificia Universidad Católica de mucosal immunity has been extensively approached, most of the studies fail in Valparaı´so, Chile addressing the mechanisms underlying their pharmacological effects. Unstandardized *Correspondence: testing as an extended practice also questions the reproducibility and safety of such Jorge Galindo-Villegas jorge.galindo-villegas@nord.no studies, limiting the use of phytogenics at commercial scale. The information presented herein provides insight on the fish mucosal immune responses to phytogenics, suggesting Specialty section: their mode of action, and ultimately encouraging the practice of reliable and reproducible This article was submitted to Comparative Immunology, research for novel feed additives for aquafeeds. For proper screening, characterization a section of the journal and optimization of their mode of action, we encourage the evaluation of purified Frontiers in Immunology compounds using in vitro systems before moving forward to in vivo trials. The Received: 15 April 2021 Accepted: 31 May 2021 formulation of additives with combinations of compounds previously characterized is Published: 18 June 2021 recommended to avoid bacterial resistance. To improve the delivery of phytogenics and Citation: overcome limitations associated to compounds volatility and susceptibility to degradation, Firmino JP, Galindo-Villegas J, the use of encapsulation is advisable. Besides, newer approaches and dedicated Reyes-López FE and Gisbert E (2021) Phytogenic Bioactive Compounds methodologies are needed to elucidate the phytogenics pharmacokinetics and mode of Shape Fish Mucosal Immunity. action in depth. Front. Immunol. 12:695973. doi: 10.3389/fimmu.2021.695973 Keywords: immunity, MALT, organosulfurs, terpenes, sustainable aquaculture, teleost, phytogenic additive, TRPV4 Frontiers in Immunology | www.frontiersin.org 1 June 2021 | Volume 12 | Article 695973 Firmino et al. Phytogenics and Fish Mucosal Immunity INTRODUCTION (12–16). Besides, the immune system influences the regulation and composition of the microbiota and vice versa, an interaction Sustainable food supply to feed the demand of the projected world that plays a determinant role in the maintenance of the mucosal population by 2050 is a challenge, in which aquaculture is predicted integrity and functionality (17, 18). Hence, both the improvement to be the main source of aquatic dietary proteins. Such growth will of the mucosal barrier characteristics and the modulation of the unavoidably involve the implementation of innovative aquaculture microbiota are central targets for the development of new production strategies, targeting issues related to effective health phytogenic additives, while understanding their mode of action management and animal welfare (1). In this regard, the at cellular and molecular levels is critical for elucidating their development and application of functional feeds represent a benefits to the host. sound strategy to improve aquaculture systems, since they provide Given the extended literature available on plant-based functional health benefits to animals beyond their nutritional value functional additives and the significance of the mucosal (2). In this scenario, phytogenics, also known as phytobiotics, are immunity described above, our review efforts focus exclusively defined as environmentally friendly plant-derived bioactive on the physiological and immunological responses achieved by the compounds used as functional feed additives that show positive most studied fish mucosal tissues, intestine, gills and skin, of effects on animal growth and health. Phytogenic often comprise organisms fed with phytogenics of the Lamiaceae family and aromatic plants extracts, and essential oils characterized by its Allium sp. In the first part, we present a thorough description richness in biologically active compounds (3, 4). In farmed fish, a on their main bioactive compounds and relevant biological wide spectrum of phytogenics have been increasingly studied properties. Then, the immunomodulatory properties and the mainly due to their wide repertoire of properties, including mechanisms they can trigger on the fish mucosal tissues are growth promotion, and antimicrobial, immunostimulant, explored and further potential mechanisms hypothesized. antioxidant, anti-inflammatory and sedative activities (5). In Finally, research gaps and constrains for the development of particular, phytogenics derived from Lamiaceae family and Allium applicable phytogenic-based additives are discussed. Overall, the sp. are among the most commonly studied and administrated plant- information presented herein aims to provide clear insights on the based additives (6, 7). Nonetheless, the complexity of the fish mucosal immune response dietary treated with phytogenics, mechanisms of action and the pharmacological effects promoted propose viable mechanisms for exploiting them, and ultimately by the diverse bioactive compounds present in such plants, along encourage the practice of reliable and reproducible research for the with their frequently observed synergistic behavior (8), often limits development of novel feed additives to be used as sustainable and the full understanding of their biological activity (9). safe prophylactic strategies for aquaculture. Since outbreaks of fish diseases are one of the main constrains for the progress of the aquaculture sector (10), the inclusion of phytogenics’ in aquafeeds is achieving significant attention at a FISH MUCOSAL IMMUNITY AT A GLANCE global scale. The impact of phytogenics upon fish systemic immunity has been extensively tested in the past (5, 9). The mucosal barrier constitutes the fish first line of defense However, an increasing trend to evaluate phytogenics’ impact against the surrounding environment and potential pathogens. upon the mucosal immunity has been gained importance in Fish mucosal tissues are particularly characterized by a mucosa- recent years, which is mainly attributed to the importance of the associated lymphoid tissue (MALT), harboring diverse myeloid mucosal barrier in the fish defense against variated threats and, and lymphoid cells that are responsible for the host protection potential colonization and invasion by pathogenic organisms against pathogens and antigens, while tolerating beneficial (11). In contrast, most of the studies evaluating the effect of symbiont colonization to maintain mucosa homeostasis (19– phytogenics in fish systemic immunity are only supported by a 21). Six different MALTs have been described so far in teleosts. selection of repetitive primary analyses (i.e., lysozyme, The gut-associated lymphoid tissue (GALT), the gill-associated bacteriolytic and complement activities, immunoglobulins, lymphoid tissue (GIALT), the skin-associated lymphoid tissue oxidative stress enzymes, etc.) serving only as proxies, that (SALT), the nasopharynx-associated lymphoid tissue (NALT) only provide a snapshot of the effects of the evaluated feed and, the more recently characterized the buccal, and pharyngeal additive on the organism. These approaches do not allow MALTs (22). Other mucosal immune systems have been elucidating their mode of action at cellular and molecular hypothesized and are currently under study (23). Despite the levels. This is of special relevance when dealing with functional existence of others, the GALT, GIALT and SALT are the most feed additives with potential pharmacological properties, as their studied and well characterized MALTs and therefore selected as standardized use mainly depends on the proper understanding of target in this review. their regulatory properties in the immune system. Among the extensive cell types with immune capacity Phytogenic administration has the potential to regulate the coexisting in the fish body, upon sensing the presence of mucosal barrier function by means of several molecular pathogenic or commensal microbe-associated molecular mechanisms, in which the phytogenic bioactive compounds pattern (MAMP) a downstream signaling response mediated interact with cellular transcription factors and metabolic through pattern recognition receptors (PRRs) immediately takes cascades. Therefore, the modulation of the expression of genes place. So far, several piscine PRRs have been identified, being the coding for immune relevant molecules alter the mucosal toll-like receptors (TLR), NOD-like receptors (NLR) and retinoic protective characteristics and their immunological status acid-inducible gene I (RIG-I)-like receptors (RLRs) the best Frontiers in Immunology | www.frontiersin.org 2 June 2021 | Volume 12 | Article 695973 Firmino et al. Phytogenics and Fish Mucosal Immunity characterized (24, 25). Epithelial and endothelial cells together The mucosal tissues are intrinsically characterized by mucus with the professional phagocytes, represented by macrophages, secreting cells, such as goblet cells (40). Beside playing important granulocytes and dendritic cells, are the first responders against roles in intra- and interspecific ecological interactions (41) and MAMPs formerly sensed by PRRs. Phagocytosis contributes to being a key component that ensures host-microbiota both pathogen clearance and subsequent antigen presentation to homeostasis (42, 43), the secreted semipermeable mucus other immune cells by the membrane Major Histocompatibility represents the first challenge that pathogens have to overcome Complex (MHC) class II peptide complex (26). In most, but not in order to interact with the host. Its complex composition all teleost fish, the peptide-MHC II complex activates naive CD4+ encompasses a matrix of glycoproteins, the mucins that confer T cells expressing antigen-specific T cell receptors (TCR) in their the mucus its structure, and a wide variety of humoral immune surface. Recognition of this complex stimulates the dedicated factors, such as lysozymes, complement, lectins, proteolytic CD4+ cells activation and differentiation into T helper cell enzymes, antimicrobial peptides, immunoglobulins, among subsets possessing inflammatory cytokines secreting capacity others (41). Moreover, the mucus is continuously secreted and that further coordinate the adaptive response together with B replaced (44); this continuous secretion aims to prevent cells (27, 28). Interestingly, while CD4+ T helper lymphocytes are pathogen attachment and interaction with the host. Therefore, mainly present in the gut lamina propria, the cytotoxic CD8+ the presence of a mucus layer is fundamentally involved in the cells are the dominant intraepithelial resident immunocytes regulation of the mucosal immune system, not only as a (29–31). protective physical and chemical barrier, but also acting as a In fact, both T and B lymphocytes are abundantly present in vehicle for mucins and humoral immune factors from the inside fish mucosal tissues (32). Interestingly, the phagocytic and out. Both goblet cells (13, 15) and mucus composition (45) are bacterial-killing abilities of B cells in fish have been fairly highly susceptible to be manipulated through dietary strategies, introduced in the past (33). However, their antigen presentation which opens a wide range of possibilities when to design and mechanisms mediated by MHC II and costimulatory molecules apply new functional feed additives. (CD80/CD86 and CD83) to prime naïve CD4+ T-cells, produce IgM, IgT, and eventually IgD plasmablasts -a major lymphocyte population in the gut, gill and at some extent the skin-, have just been recently addressed (34, 35). The IgT, the teleost specialized LAMIACEAE FAMILY AND ALLIUM SP. mucosal immunoglobulin analogous to mammalian IgA (36), AMONG THE MOST STUDIED plays a critical role in the clearance of mucosal pathogens and the AROMATIC PLANTS USED AS preservation of microbiota homeostasis through immune PHYTOGENICS IN AQUACULTURE exclusion (11). Although extremely important in mucosal defense, not all teleosts present IgT/IgD, which suggests the In nature, plant secondary metabolites have functional roles existence of alternative mucosal immune systems (23). independent from plant growth and development; thus, For instance, the complete IgM and IgT sequences in their protecting plants from herbivore and pests, or acting as membrane and soluble forms have been reported and characterized chemoattractants for pollinators (46). These bioactive for the first time in a perciform, the gilthead sea bream (Sparus compounds broadly found in aromatic plants are usually aurata) (37). Interestingly, this study demonstrated that virus and present as mixtures, mainly represented by phenolics and bacteria trigger the mucosal immune response by promoting the terpenes that are chemically characterized by their aromatic activation of IgT in seabream. Although, diets with fish oil rings (3). Therefore, their benefits as dietary supplements are replacement by ones from plant origin inhibited the IgT up- subject to the variability and complexity of the aromatic regulation upon intestinal parasitic challenge, which was related compounds mixture, apart from their synergistic effect, their to a worse disease outcome. These results evidenced that mucosal origin, the dietary inclusion level and their pharmokinetics (47). immunoglobulins can be significantly affected by dietary treatments, In particular, phytogenics derived from Lamiaceae family and which highlights the necessity of testing this response case by case. Allium sp. are among the most widespread administrated plant- Although characterized by common cellular components, based additives in aquaculture (48) and livestock (6, 7). These immune mediators and immune mechanisms, the different compounds are used for their recognized growth promoting, composition, organization and functions of MALTs may vary antimicrobial, immunostimulant, antioxidant, anti-inflammatory according to each tissue intrinsic and external environmental and sedative properties. Although they can be found worldwide, factors (38), changes that may be associated to the fish species some representatives of this group of aromatic plants (i.e., oregano, considered. Besides, the microbiota also stands as a relevant thyme, basil, menthe, rosemary, sage, marjoram, garlic and onion, component of the mucosal barrier, displaying an antagonistic among others) are particularly present and traditionally consumed behavior against invading “hostile” microorganisms and directly in the Mediterranean area and appreciated in terms of human participating in the immune responses through the complex nutrition and therapy (49, 50). The health-promoting properties of host-microbiota crosstalk at the mucosal interface (17, 18). these aromatic plants have been extensively reviewed in different Therefore, the selective manipulation of the microbiota by aquaculture species (5, 48, 51–55). However, most of the studies means of nutritional approaches has been previously proposed dealing with these functional feed additives were only focused in as a viable alternative to modulate mucosal responses (39). physiological or biochemical responses, but few of them have Frontiers in Immunology | www.frontiersin.org 3 June 2021 | Volume 12 | Article 695973 Firmino et al. Phytogenics and Fish Mucosal Immunity elucidated the cellular and molecular mechanisms underlying their Together with their well-studied bactericidal potential, these immunostimulatory capacity. phenolic compounds are described to potentially improve the While the existent information about the inherent effect of integrity of the mucosal tissues due to their observed antioxidant, these phytogenics upon immune cells is limited under in vitro anti-inflammatory and consequent immunomodulatory conditions, numerous in vivo studies have demonstrated an properties in the gastrointestinal mucosa of several animal improvement of the fish mucosal immune responses following models (112). The reported strong antioxidant activity of their administration. A refined complementary search through carvacrol and thymol rely on their ability to scavenge free Web of Science, PubMed and Google Scholar was performed in radicals, inhibiting reactive oxygen species (ROS) and other this review. Until March 2021, 62 publications reporting the oxygen radicals generated in cells and tissues (113). By nutritional effects of Lamiaceae family and Allium sp., or related contrast, high concentrations may display antagonistic pro- bioactive compounds, upon fish mucosal responses were retrieved oxidant effects (113). This dose-dependent antagonistic activity and their results summarized in Table 1. Importantly, most of evidences the importance of correctly define their administration them were published in the last year; thus, evidencing the current doses in order to obtain the desired results with regard to their increasing interest for research on phytogenics targeting mucosal immunomodulatory properties. tissues. From the overall bibliographic search results, few Regarding their anti-inflammatory potential, carvacrol and publications felt within the objective of the present review and thymol appear to interfere with the NF-kB and MAPK pathways, described the cellular or molecular mechanisms underlying fish modulating the expression of pro-inflammatory and anti- mucosal immune responses to phytogenics’ administration. inflammatory cytokines (114, 115). It is commonly speculated Studies reporting the application of plant extracts or related that the anti-inflammatory properties of plant-derived bioactive compounds as bath treatments or evaluating bactericidal or compounds, such as carvacrol and thymol, may be attributed to antiparasitic effects in vitro were excluded from the selection as their capacity to inhibit TLR-mediated NF-kB signaling pathways this review is just focused on the mucosal immune mechanisms. (116, 117). Furthermore, evidence that both carvacrol and thymol Furthermore, Table 1 omits those results out of the mucosal play a role in the chemosensory system through the activation of immunity context, including systemic immunity-related results, transient receptor potential (TRP) cation channels exist (118, 119). non-immune digestive parameters or other complementary In higher vertebrates, TRP channels are widely expressed in several analysis performed within each study. Although such variables cellular types that includes most of the mucosal components. are extensively used as key performance indicators in such studies, Through the maintenance of the intracellular calcium their relevance in terms of supporting and/or establishing the homeostasis, these channels are known to regulate several cell mode of action of phytogenics is questionable and out of the scope functions, such as stimuli perception, inflammatory molecules for this review. Finally, blends with other components besides the production and secretion, migration and even phagocytosis (120– selected group of plants –or associated bioactive compounds such 122). Carvacrol and thymol are known to activate both the receptor as terpenes or organosulfurs– were excluded as well. TRPA1 (119) and the receptor TRPV3 in mucosal tissues, elevating cytosolic Ca2+ concentration in epithelial cells (118, 123). In fish, Effect of Dietary Terpene Phenolic together with TLRs, the activation of TRP channels has been Compounds Upon Fish Mucosal Immunity demonstrated to modulate the inflammatory processes through Phenolics and terpenes are a group of volatile plant-derived the activation of the TRP/Ca2+/TAK1/NF-kB signaling pathway bioactive compounds with medicinal and biotechnological value (124). This suggests that a TRP channel mediated cellular activation that constitute the dominant fraction of the essential oils derived may underlie the immunomodulatory properties of these from aromatic plants (3). The monoterpenes carvacrol and its bioactive compounds. isomer thymol are the most studied phenolic compounds, The health promoting effects of oregano, thyme and their representing the major components of the essential oils from derivates in fish have been recently reviewed (55, 125). several aromatic plants of the Lamiaceae family like the oregano Concerning their impact upon fish mucosal immunity, several (Origanum vulgare) and thyme (Thymus vulgaris) (109, 110). These nutritional studies have described beneficial effects of compounds are particularly studied and recognized for their phytogenics derived from oregano, thyme and other plants of bactericidal activity, since their lipophilic character act as bacterial the Lamiaceae family upon the mucosal tissues in several fish membrane permeabilizers with cytotoxic effects upon bacterial species (Table 1). Most of them have reported an increase in skin structure and function, leading to membrane expansion, fluidity mucus immune markers and/or skin mucus bactericidal activity and permeability, disturbance of the membrane-embedded (56–58, 65, 71, 73–79). The repeatedly evaluated markers were proteins, respiration inhibition and alteration of ion transport. In lysozyme, alkaline phosphatase, complement and protease addition, carvacrol and thymol were demonstrated to act as quorum activities, total immunoglobulin and protein content in fish sensing (QS) inhibitors, reducing bacterial biofilm formation. skin mucus, as well as its in vitro bactericidal potential against Carvacrol in particular, is able to inhibit bacteria motility, bacterial pathogens. Several of these studies also described an collapsing the proton-motive force, depleting the ATP pools and improvement in key performance indicators, such growth, feed preventing the synthesis of flagellin (111). This bactericidal property efficiency and survival against pathogenic bacterial challenges highlights the ability of these compounds to potentially modulate (56, 58, 65, 71, 73–79). Besides the assessment of key mucosal tissues associated microbiota. performance indicators and general immune markers in skin Frontiers in Immunology | www.frontiersin.org 4 June 2021 | Volume 12 | Article 695973 TABLE 1 | Extended summary of the current available literature on nutritional effects of Lamiaceae family and Allium sp. derived phytogenics upon fish mucosal immune response. Frontiers in Immunology | www.frontiersin.org Firmino et al. Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference plant origin form dosage(s) administration components (≤ 3) tested Lamiaceae family Oregano Powder 0.5%, 8 weeks N/I Zebrafish (Danio ↑ Final weight ↑ Skin mucus lysozyme activity Beneficially affects the skin mucus Rashidian, Boldaji (Origanum 1.0% and rerio) ↑ Weight gain ↑ Skin mucus alkaline phosphatase immune parameters, growth (56) vulgare) 2.0% ↓ FCR activity performance and survival against ↑ SGR ↑ Skin mucus total Ig pathogenic bacterial challenge ↑ Survival ↑ Skin mucus protease activity against A. ↑ Skin mucus total protein hydrophila Oregano Powder 0.5% and 15 and N/I Gilthead seabream No effect ↑ Skin mucus IgM Oregano improves humoral immunity Beltrá n, Gonzalez (Origanum 1.0% 30 days (Sparus aurata) ↑ Skin mucus bactericidal activity and increases the antibacterial activity Silvera (57) vulgare) against P. damselae of skin mucus Oregano Ethanolic extract 0.2% and 60 days + 7 N/I Nile tilapia ↑ Final weight ↑ Skin mucus total Ig Can effectively improve the fish Mohammadi, (Origanum 0.5% days A. (Oreochromis ↑ Weight gain growth, health, and immune status Rafiee (58) vulgare) hydrophila niloticus) ↓ FCR challenge ↑ SGR ↑ Survival rate ↑ Survival against A. hydrophila Oregano Powder 5.0, 10.0, 15.0 8 weeks Carvacrol and thymol Common carp ↑ Final weight ↑ Intestine villus height Dose-dependent enhancement of Abdel-Latif, Abdel- (Origanum and 20.0 g kg- (Commercial product) (Cyprinus carpio) ↑ Weight gain ↑ Intestine villus width intestinal morphometry, which Tawwab (59) vulgare) 1 ↑ SGR ↑ Intestine crypt depth subsequently lead to improvement of nutrients absorption Oregano Essential oils 0.75, 1.5, 2.25 64 days Carvacrol, thymol and Nile tilapia No effect ↑ Intestine villus height Increases intestinal villus size Heluy, Ramos (60) (Origanum and 3.0 g kg-1 p-cymene (Oreochromis vulgare) niloticus) Oregano Essential oils 0.5, 1.5 and 8 weeks + 7 N/I Koi carp (Cyprinus ↑ Survival ↓ TNF-a and TGF-b gene expression Presents immunomodulatory effects Zhang, Wang (61) (Origanum 4.5 g kg-1 days A. carpio) against A. in intestine and enhances disease resistance. vulgare) hydrophila hydrophila ↑ Actinobacteria phylum, and Also beneficially alters the gut bacterial challenge Propionibacterium, Brevinema and community composition of fish 5 Corynebacterium genera ↓ Bacteroidetes phylum and Vibrio genus No effect on bacterial alpha diversity Oregano Powder 0.5% and 30 days + Carvacrol and thymol Common carp N/I ↓ Gill histopathologic lesions Protective roles against the adverse Khafaga, Naiel (62) (Origanum 1.0% cypermethrin (Commercial product) (Cyprinus carpio) ↓ Gill proliferating cell nuclear antigen effects of cypermethrin, enhancing vulgare) exposure (PCNA) and caspase-3 immune recovery from the exposure positive cells Oregano Essential oils 0.01%, 0.02%, 24 days + 28 Carvacrol, p‐cymene and g‐ Chum salmon ↑ feed efficiency Carvacrol content detected in the skin Preventive effects against I. salmonis Mizuno, Urawa (Origanum 0.05% and days I. salmonis terpinene (Oncorhynchus keta) ↓ I. salmonis of fish fed the oregano supplemented and T. truttae and suggests the (63) vulgare) 0.10% and T. truttae infection diet possibility that its anti‐parasitic action challenge ↓ T. truttae is attributable to the bioactive (Total 52 days) infection component emergence through the ↓ cumulative skin mortality Oregano Essential oils 0.5, 1.0, 1.5, 90 days N/I Yellow N/I ↑ Intestine villus length Promotes increased absorption Ferreira, Caldas (Origanum 2.0 (Commercial product) tail tetra (Astyanax ↑ Intestine villus width surface area and modulates the (64) vulgare) and 2.5 g kg-1 altiparanae) ↑ Intestine absorption area number of goblet cells involved in ↑↓ Intestine goblet cells number June 2021 | Volume 12 | Article 695973 protecting the intestinal mucosa Phytogenics and Fish Mucosal Immunity Marjoram Ethanolic extract 0.1, 0.2 and 60 days + 10 N/I Common carp ↑ Final weight ↑ Skin mucus alkaline phosphatase Increase fish skin mucosal immunity Yousefi, (Origanum 0.4 g kg-1 days A. (Cyprinus carpio) ↑ Weight gain ↑ Skin mucus total Ig and performance Ghafarifarsani (65) majorana) hydrophila ↓ FCR ↑ Skin mucus lysozyme activity challenge ↑ SGR ↑ Skin mucus alternative complement ↑ Survival (ACH50) activity against A. hydrophila Thyme Essential oils 500 ppm 30 days + Thymol, p-cymene and g- African catfish N/I ↓ Gill histopathologic lesions Mitigate the thiamethoxam induced El Euony, Elblehi (Thymus thiamethoxam terpinene (Clarias garipenus) toxicity (66) vulgaris) exposure Thyme Aqueous extract 5.0, 10.0 and 2 weeks + N/I Rainbow trout No effect ↑ Intestine antioxidant enzymes (SOD, Mitigate adverse effects of Hoseini and (Thymus 20.0 g kg-1 oxytetracycline (Oncorhynchus CAT, GPx and GST) activity oxytetracycline and improve the fish Yousefi (67) vulgaris) mykiss) ↓ Intestine levels of the oxidative immune responses stress marker malondialdehyde (Continued) Frontiers in Immunology | www.frontiersin.org Firmino et al. TABLE 1 | Continued Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference plant origin form dosage(s) administration components (≤ 3) tested Thyme Essential oils 0.1%, 0.5%, 15 days Thymol, o-cymene and carvacrol Nile tilapia N/I No effect upon the population of Stimulated the cellular components of Valladão, Gallani (Thymus and 1% (Oreochromis beneficial Bacillus bacteria in the gut the non-specific immune response (68) vulgaris) niloticus) without deleterious effects on the general health of the fish or the intestinal tract Thyme Essential oils 0.005, 0.010 5 weeks Thymol, p‐cymene and Rainbow trout No effect No effect upon the allochthonous No toxic effects do not significantly Navarrete, Toledo (Thymus and 0.02 g linalool (Oncorhynchus microbiota profile alter the intestinal contents bacterial (69) vulgaris) kg-1 mykiss) populations Spanish Essential oils 0.001, 0.002, 12 weeks Thymol, p‐cymene and carvacrol Gilthead seabream No effect ↑ Anterior intestine lymphocyte Dose-dependent immuno‐modulatory Hernandez, Garcia thyme 0.003 and (Sparus aurata) aggregates in the lamina propria at effect upon the intestine (70) (Thymus 0.004 g kg-1 low dose zygis subsp. ↓ Anterior intestine lymphocyte gracilis) aggregates in the lamina propria at high doses Lemon balm Ethanolic extract 0.2% and 60 days + 7 N/I Nile tilapia ↑ Final weight ↑ Skin mucus total Ig Can effectively improve the fish Mohammadi, (Melissa 0.5% days A. (Oreochromis ↑ Weight gain ↑ Skin mucus lysozyme activity growth, health, and immune status Rafiee (58) officinalis) hydrophila niloticus) ↓ FCR ↑ Skin mucus protease activity challenge ↑ SGR ↑ Skin mucus alternative complement ↑ Survival rate (ACH50) activity ↑ Survival against A. hydrophila Peppermint Powder 2.0, 3.0, and 8 weeks N/I Caspian roach ↑ Final weight ↑ Secretion of skin mucosal protein Act as a growth promoter and Paknejad, Hosseini (Mentha 4.0 g kg-1 (Rutilus caspicus) ↑ Weight gain pattern bands; higher lysozyme band immunostimulant Shekarabi (71) piperita) ↓ FCR intensity in particular ↑ SGR ↑ Skin mucus lysozyme activity ↓ Daily intake ↑ Skin mucus alkaline phosphatase rate activity ↑ Skin mucus soluble protein Peppermint Essential oils 0.1 and 0.25 g 7, 14, 30 and Menthol, mentone and 1,8- Nile tilapia N/I ↑ Intestine intraepithelial lymphocytes Show benefits in terms of intestinal Valladão, Gallani 6 (Mentha kg-1 60 days cineole (Oreochromis health and on immune parameters (72) piperita) niloticus) Peppermint Ethanolic extract 1.0%, 2.0% 8 weeks N/I Rainbow trout ↑ Survival ↑ Skin mucus antibacterial activity Triggers the immune system of Adel, Pourgholam (Mentha and 3.0% (Onchorhynchus against A. against S. iniae, Y. ruckeri, A. rainbow trout against Y. ruckeri (73) piperita) mykiss) hydrophila hydrophila and L. garviea Peppermint Ethanolic extract 1.0%, 2.0% 56 days N/I Caspian kutum ↑ Weight gain ↑ Skin mucus antibacterial activity Increases the mucosal immune Adel, Amiri (74) (Mentha and 3.0% roach (Rutilus frisii ↓ FCR against S. iniae, Y. ruckeri, L. parameters and performance of fry in piperita) kutum) ↑ SGR monocytogenes and E. coli a dose dependent manner ↑ Skin mucus protein level ↑ Skin mucus alkaline phosphatase activity Peppermint Ethanolic extract 1.0%, 2.0% 8 weeks N/I Caspian brown trout ↑ Weight gain ↑ Skin mucus protein level Promote growth performance and Adel, Safari (75) (Mentha and 3.0% (Salmo trutta ↓ FCR ↑ Skin mucus lysozyme activity have immunostimulant properties piperita) caspius) ↑ SGR ↑ Skin mucus alkaline phosphatase activity Horsemint Ethanolic extract 2.0%, 4.0% 8 weeks + Y. N/I Caspian kutum ↑ Weight gain ↑ Skin mucus protein level Improve growth performance and Gholamhosseini, (Mentha and 6.0% ruckeri challenge roach (Rutilus frisii ↓ FCR ↑ Skin mucus lysozyme activity boost fish immune response in a Adel (76) longifolia) kutum) ↑ SGR ↑ Skin mucus alkaline phosphatase dose‐related manner June 2021 | Volume 12 | Article 695973 ↑ Survival rate activity Phytogenics and Fish Mucosal Immunity ↑ Skin mucus protease activity ↑ Skin mucus esterase activity ↑ Skin mucus antibacterial activity against S. iniae, Y. ruckeri, A. hydrophila and L. garvieae Horsemint Hydroalcoholic 0.1%, 0.2% 4 weeks + 10 N/I Rainbow trout ↑ Survival ↑ Secretion of skin mucosal protein Dose‐related positive effect on Heydari, (Mentha extract and 0.3% days Y. ruckeri (Onchorhynchus against Y. pattern bands; higher lysozyme band immunogenicity and increased Firouzbakhsh (77) longifolia) challenge mykiss) ruckeri intensity in particular resistance to bacterial disease Thumbai Powder 1.0, 2.0, 4.0 45 days + 15 N/I Nile tilapia ↑ Weight gain ↑ Skin mucus lysozyme activity Increase skin mucosal immune Kurian, Van Doan (Leucas and days S. (Oreochromis ↓ FCR ↑ Skin mucus peroxidase activity parameters, performance and survival (78) aspera) 8.0 g kg-1 agalactiae niloticus) ↑ SGR against bacterial infection challenge ↑ Survival (Continued) Frontiers in Immunology | www.frontiersin.org Firmino et al. TABLE 1 | Continued Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference plant origin form dosage(s) administration components (≤ 3) tested against S. agalactiae Shirazi Hydroalcoholic 2.0 g kg -1 56 days Thymol and carvacrol? (N/I) Rainbow trout ↑ Survival rate ↑ Skin mucus bactericidal activity Increase skin mucosal immunity Mirghaed, Hoseini thyme extract (Oncorhynchus against A. hydrophila (79) (Zataria mykiss) ↑ Skin mucus lysozyme activity multiflora) Shirazi Powder (1:1) 40 g kg-1 12 weeks N/I Common carp No effect No effect Do not prevent intestinal tissue lesions Tasa, Imani (80) thyme + aflatoxin B1 (20 g kg-1 (Cyprinus carpio) induced by aflatoxin B1 (Zataria each) multiflora) + Rosemary (Rosmarinus officinalis) Rosemary Aqueous extract 10, 20, 40, 80 20 days 1,8-Cineole Common carp N/I ↑ Skin mucus level of 1,8-Cineole High volume of extracts might Zoral, Ishikawa (Rosmarinus and 100 ml/ (Cyprinus carpio) dose-dependent promote hepatic toxicity (81) officinalis) 100 g No effect upon intestine histopathology Rosemary Powder 0.6, 1.2, 1.8 4 and 12 weeks Carnosic acid and carnosol (1:1) Gilthead seabream No effect No effect The histological examination of the Hernandez, Garcia (Rosmarinus and 2.4 g kg-1 (Sparus aurata) intestine showed no aspects that Garcia (82) officinalis) might pose problems for absorption, or any immune system disorder associated with the intestine Oliveria Essential oils and/ 0.01%, 0.1% 60 days + 14 g-terpinene, Nile tilapia ↑↓ Survival No effect Increase fish survival 14 days after Vazirzadeh, Jalali (Oliveria or hydroethanolic and 1.0% days S. iniae carvacrol and thymol (Oreochromis against S. iniae challenge with S. iniae (83) decumbens) extract challenge niloticus) Clove basil Ethanolic extract 5.0, 10.0, and 12 weeks + 14 N/I African catfish ↑ final weight ↑ intestine villus length Improve the fish performance, health, Abdel-Tawwab, (Ocimum 15.0 g kg-1 days L. (Clarias gariepinus) ↑ weight gain ↑ intestine villus width and immune response Adeshina (84) gratissimum) monocytogenes ↑ SGR ↑ intestine absorption area challenge ↑ Feed intake ↑ survival 7 against L. monocytogenes Clove basil Essential oils 0.5%, 1.0% 55 days + 10 1,8-cineole, eugenol and b- Nile tilapia N/I ↑ Intestine villus height Ameliorate tissue damages, even in Brum, Cardoso (Ocimum and 1.5% days S. selinene (Oreochromis ↑ Intestine goblet cells number situations of infection (85) gratissimum) agalactiae niloticus) ↓ Gill epithelial detachment in the challenge secondary lamellae ↓ Gill congestion at the base of the secondary lamellae American Essential oils 0.25, 0.5, 1.0 7 weeks Linalool, eugenol and 1,8-cineole Red drum No effect ↑ Stomach lysozyme activity Different supplementation levels do not Sutili, Velasquez basil and 2.0 g kg-1 (Sciaenops No effect upon the intestinal microbial influence growth performance and (86) (Ocimum ocellatus) community intestinal microbial community; americanum) however, show effects on immunological responses Savory Powder 1% 45 days N/I Common carp N/I ↑ Intestinal lactic acid bacteria Improves intestinal health Mousavi, (Satureja (Cyprinus carpio) Mohammadiazarm khuzestanica) (87) Allium sp. Garlic Aqueous extract 0.10, 0.15, and 80 days N/I Guppy (Poecilia No effect ↑ Skin mucus lysozyme Administration of 0.15 mL of garlic Motlag, Safari (88) June 2021 | Volume 12 | Article 695973 (Allium 0.20 ml kg-1 reticulata) ↑ Skin mucus alternative complement extract per kg feed is suggested to Phytogenics and Fish Mucosal Immunity sativum) ↑ Skin mucus total Ig obtain optimal skin mucus immunity ↑ Skin mucus alkaline phosphatase Garlic Oil 50 µl kg-1 28 days + N/I Rohu (Labeo rohita) N/I ↓ Gill oxidative stress enzymes activity Amelioration of silver nanoparticles- Khan, Qureshi (89) (Allium exposure to ↓ Gill histopathologic lesions induced oxidative stress and sativum) silver histoprotective effects nanoparticles Garlic Powder 0.5 g and 1.0 2 months + N/I Nile tilapia ↑ Survival ↑ Anterior intestine transcriptional Could be effective in the prevention of Foysal, Alam (90) (Allium g/100 g 2 weeks S. iniae (Oreochromis against S. iniae levels of interleukin genes (IL‐10 and S. iniae infection in fish sativum) challenge niloticus) IL‐17F) ↑ OTU counts for the phylum of Proteobacteria and Tenericutes Garlic Powder 5.0%, 10.0% 14 or 28 or 32 Allicin (1.25 mg/g) Guppy (Poecilia ↓↑ Gills and N/I No clear preventative effect against C. Kim, Fridman (91) (Allium and 20.0% days + C. reticulata) caudal fin C. irritans sativum) irritans challenge irritans infection (Continued) Frontiers in Immunology | www.frontiersin.org Firmino et al. TABLE 1 | Continued Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference plant origin form dosage(s) administration components (≤ 3) tested Garlic Powder 1.0%, 1.5% 120 days Rainbow trout ↑ Weight gain ↓ Bacterial diversity and richness Beneficial in terms of promoting Büyükdeveci, (Allium and 2.0% (Oncorhynchus ↑ SGR ↑Deefgea, Mycoplasma, growth and inducing changes in the Balcá zar (92) sativum) mykiss) Exiguobacterium and Clostridium intestinal microbiota in a dose- genera dependent manner ↓↑ Aeromonas genus Garlic Powder 5.0, 10.0 and 8 weeks N/I Caspian roach ↑ Weight ↑ Skin mucus antibacterial activity Beneficially affects the skin mucus Ghehdarijani, (Allium 15.0 g kg-1 (Rutilus rutilus) ↑ Growth rate against S. faecium, M. luteus, S. immune parameters and growth Hajimoradloo (93) sativum) diet marcescens and E. coli performance ↑ Skin mucus protein level ↑ Skin mucus alkaline phosphatase activity Garlic Lyophilized 2.0% 21 days + N/I Prussian carp N/I ↓ Gill histopathologic lesions Shows chelating and antioxidant Nicula, Dumitrescu (Allium cadmium (Carassius gibelio) potential (94) sativum) exposure Onion (Allium Powder 1% 45 days N/I Common carp N/I ↑ Intestinal lactic acid bacteria Improves intestinal health Mousavi, cepa) (Cyprinus carpio) Mohammadiazarm (87) Onion (Allium Ethanolic extract 0.5%, 1.0%, 12 weeks N/I African sharptooth No effect ↓ Intestine villus length Increase the digestive and absorptive Bello, Emikpe (95) cepa) 1.5% and catfish (Clarias ↑ Intestine villus width capacity of the intestine 2.0% gariepinus) ↑ Intestine absorption area ↑ Intestine cryptal depth Mongolian Ethanolic extract 0.04 g kg-1 4 weeks + Flavonoids ≥90% (HPLC) Grass carp N/I ↓ Intestine and gill Cr accumulation Decrease in Cr-accumulation, Zhao, Yuan (96) Wild Onion chromium (Cr) (Ctenopharyngodon ↓ Gill malondialdehyde content oxidative stress, immunosuppression (Allium exposure idella) ↓ Gill protein carbonyl content and inflammatory response following mongolicum) ↑ Intestine lysozyme activity Cr exposure ↑ Intestine complement 3 levels ↑ Intestine and gill tight junction proteins gene expression ↑↓ Intestine and gill NF-kB signaling pathway gene expression 8 Single bioactive compounds Thyme Ethanolic solution 0.15, 0.3, 0.45, 56 days + 14 Thymol (commercial product) Snakehead fish ↑ Final weight ↑ Intestine SOD, CAT, GSH-Px Adequate dietary supplementation can Kong, Li (97) (Thymus 0.6, 0.75 g kg- days A. veronii (Channa argus) ↑ Weight gain activities effectively enhance the growth, vulgaris)* 1 challenge ↑ SGR ↓ Intestine malondialdehyde content antioxidant status, immune response ↑ Protein ↑ IL-10 and TGF-b gene expression in and disease resistance efficiency ratio intestine ↓ FCR ↓ HSP70, TNF-a, IL-1b and IL-8 gene ↑ Survival to A. expression in intestine veronii Thyme N/I 0.1, 0.2 and 60 days + 8 Thymol (commercial product) Grass carp ↑ Final weight ↑ Gill enzymes of the phosphotransfer Favors weight gain and fish longevity. Morselli, Baldissera (Thymus 0.3 g kg-1 days A. (Ctenopharyngodon ↑ Survival network: cytosolic and mitochondrial Prevents A. hydrophila induced (98) vulgaris)* hydrophila idella) against A. creatine kinases, adenylate kinase branchial bioenergetics. High challenge hydrophila activities and ATP levels in infected concentrations deserve attention fish because of side-effects ↓ Gill ROS levels in infected fish Thyme Crystals 0.5 g kg-1 70 days Thymol (99% purity; commercial Nile tilapia ↑ SGR ↓ Intestine total aerobic and anaerobic Improve some performance El-Naby, Al- (Thymus product) (Oreochromis ↑ Protein counts parameters and negatively modulates Sagheer (99) vulgaris)* niloticus) efficiency ratio intestinal microbial communities. June 2021 | Volume 12 | Article 695973 Demonstrates a notable synergistic Phytogenics and Fish Mucosal Immunity interaction with chitosan nanoparticle with beneficial effects Thyme N/I 1.0 g kg-1 56 days Thymol (commercial product) Rainbow trout ↓ FCR ↓ Intestine culturable anaerobe Modulated intestinal microbial Giannenas, (Thymus (Oncorhynchus bacteria communities disfavoring total Triantafillou (100) vulgaris)* mykiss) anaerobes Oregano N/I 1.0 g kg-1 56 days Carvacrol (commercial product) Rainbow trout ↓ FCR ↓ Intestine culturable anaerobe Modulated intestinal microbial Giannenas, (Origanum (Oncorhynchus bacteria communities disfavoring total Triantafillou (100) vulgare)* mykiss) anaerobes Chinese Powder 0.4, 0.8 and 60 days + H2O2 Baicalin (80% purity, commercial Nile tilapia ↓ FCR ↑ Gill glutathione level Improves feed efficiency, enhance Jia, Du (101) skullcap 1.6 g kg-1 challenge product) (Oreochromis ↑ Gill total antioxidant capacity antioxidative ability and alleviate (Scutellaria niloticus) oxidative stress baicalensis)* Garlic Liquid 0.005%, 30 days Allicin (98% purity, commercial Large yellow ↑ Final weight ↑ Intestine total antioxidant capacity Improve the survival and growth of Huang, Yao (102) (Allium 0.01% and product) croaker ↑ Final length ↑ Intestine antioxidant enzymes (CAT, large yellow croaker larvae probably sativum)* 0.02% NO and NOS) activity by promoting intestinal development, (Continued) Frontiers in Immunology | www.frontiersin.org Firmino et al. TABLE 1 | Continued Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference plant origin form dosage(s) administration components (≤ 3) tested (Larimichthys ↑ SGR ↓ Intestine transcriptional levels of pro- alleviating inflammation and enhancing crocea) ↑ survival rate inflammatory genes appetite Phytogenics combinations Phytogenics Essential oils 200 ppm 70 days + 15 Garlic and Lamiaceae-plants oils Greater amberjack No effect ↑ Skin mucus lysozyme activity 15 Facilitates the immunological response Ferná ndez- combination days N. girellae (N/I; commercial additive) (Seriola dumerili) days post N. girellae challenge of skin once the parasite is fixed, Montero, challenge ↑ Piscidin gene expression in skin pre- generating a hostile microenvironment Torrecillas (16) challenge in skin and lowering the parasite load ↑ Proinflammatory cytokines (tnf-a and il1-b), AMPs (hep and cath), immunoglobulin (IgT), complement protein (c3) T-cells marker (cd8) and mucin (muc-2) gene expression in skin post-challenge ↓ Casp3 gene expression in skin post- challenge Phytogenics Essential oils 0.3 g kg-1 8 weeks + 2 Cinnamaldehyde, thymol and Nile tilapia ↓ ↑ Intestine villi density Positive effects of digestion and Ning, Zhang (103) combination weeks hypoxia carvacrol (Oreochromis Hepatosomatic ↓ Intestine malondialdehyde content antioxidative capacity challenge niloticus) index (Total 10 weeks) Phytogenics Microencapsulated 0.5% 65 days Garlic essential oil (N/I), carvacrol Gilthead seabream No effect ↑ Skin mucus inhibitory activity against Beneficially affects the skin and mucus Firmino, combination essential oils and thymol (Sparus aurata) V. anguillarum and P. anguilliseptica immune and stress parameters, Ferná ndez-Alacid (Commercial additive) ↓ Cortisol in skin mucus suggesting the stimulation and (14) ↑ Regulation of genes associated to recruitment of phagocytic cells and a the secretory pathway in skin reduction in the fish allostatic load ↑ Regulation of genes associated to non-specific immune response in skin ↑ Regulation of genes coding for oxidative stress enzymes in skin Phytogenics Microencapsulated 0.5% 65 days Garlic essential oil (N/I), carvacrol Gilthead seabream No effect ↑ Regulation of genes related to The activation of leukocytes and Firmino, Vallejos- combination essential oils and thymol (Sparus aurata) processes of proteolysis and crosstalk between gut and microbiota Vidal (12) 9 (Commercial additive) inflammatory modulation, immunity, are suggested to regulate the transport and secretion, response to inflammatory response induced by the cyclic compounds, symbiosis, and additive RNA metabolism in the mid-anterior intestine No effect upon alpha diversity of bacterial communities in the anterior and posterior intestinal tract sections ↓ Spirochaetes phylum in the posterior intestine ↑ Photobacterium and Corynebacterium genera in the anterior intestine ↓ Comamonas in the anterior intestine, and Paracoccus, Prevotella and Rothia genera in the posterior intestine ↓ Bacterial sequences related to carbohydrate and drug metabolisms, June 2021 | Volume 12 | Article 695973 and membrane transport Phytogenics and Fish Mucosal Immunity ↑ Bacterial sequences related to glutathione and lipid metabolisms, naphthalene degradation and sulphur relay system Evidence of host-microbial co- metabolism Phytogenics Microencapsulated 0.5% 65 days + 39 Garlic essential oil (N/I), carvacrol Gilthead seabream ↓ S. ↑ Regulation of genes related pro- Promotes gill mucosal immunity and Firmino, Vallejos- combination essential oils days S. and thymol (Sparus aurata) chrysophrii total inflammatory immune response reduces gill ectoparasite incidence Vidal (15) chrysophrii (Commercial additive) parasitation arbitrated by degranulating acidophilic granulocytes, sustained by antioxidant (Continued) Frontiers in Immunology | www.frontiersin.org Firmino et al. TABLE 1 | Continued Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference plant origin form dosage(s) administration components (≤ 3) tested challenge (total and anti-inflammatory responses in 104 days) gills ↑ Carboxylate glycoproteins containing sialic acid in mucous and epithelial gill’s cells Phytogenics Essential oils 0.02% 9 weeks + 1 Garlic and Lamiaceae-plants oils European sea bass ↑ Survival ↑ Skin mucus lysozyme activity when Attenuate the fish physiological Serradell, combination week stress and (N/I; commercial additive) (Dicentrarchus against Vibrio stress and bacterial challenge response to stress increasing Torrecillas (104) V. anguillarum labrax) anguillarum resistance to Vibrio anguillarum challenge when stress- infection challenged Phytogenics Essential oils 0.02% 63 days Garlic and Lamiaceae-plants oils European sea bass No effect ↓ Shannon alpha diversity of mucosa- Reduction of orders containing Rimoldi, Torrecillas combination (N/I; commercial additive) (Dicentrarchus associated microbiota potentially pathogenic species for fish, (105) labrax) ↑Clostridiales order in intestinal and enrichment of gut microbiota content composition with butyrate producer ↓ Coliforms and Vibrionales taxa allochthonous microbiota Phytogenics Essential oils 200 ppm 63 days + 7 Garlic and Lamiaceae-plants oils European sea bass No effect ↓ Posterior intestine fold area covered Protective effect focused mainly on the Torrecillas, Terova combination days stress and (N/I; commercial additive) (Dicentrarchus by goblet cells preileorectal valve region (13) V. anguillarum labrax) ↓ Posterior intestine goblet cells area challenge ↑ Intestine mucus coverage post- challenge Phytogenics N/I extract 6.0 g kg-1 30 days + 10 Saint John’s wort (Hypericum Atlantic salmon No effect ↓ Gut lipid peroxidation Improves the gut antioxidant status Reyes-Cerpa, combination days crowding perforatum, Hypericacea), lemon (Salmo salar) Vallejos-Vidal (106) stress (40 days balm (Melissa officinalis, total) Lamiaceae) and rosemary (Rosmarinus officinalis, Lamiaceae) mixed at a ratio 3:2:1 Phytogenics Powder 1% 45 days Savory (Satureja khuzestanica, Common carp N/I ↑ Intestinal lactic acid bacteria Improves intestinal health Mousavi, combination Lamiaceae) 0.5% and Onion (Cyprinus carpio) Mohammadiazarm (Allium cepa, Alliaceae) 0.5% (87) 10 Phytogenics Essential oils 0.06, 0.2, 0.4 6 weeks Thymol and carvacrol (1:1; Hybrid tilapia (O. N/I ↑ Posterior intestine villus height Affect the immunity primarily through a Ran, Hu (107) combination and 0.8 g kg-1 commercial additive) niloticus ♀ × O. ↑ Posterior intestine goblet cell count direct effect on host tissue but also aureus ♂) per villus has an indirect effect mediated by ↑ Anterior intestine intraepithelial microbial changes leucocytes ↓ Distal intestine intraepithelial leucocytes ↑ OTUs, and PD whole tree and Chao1 diversity indexes ↓Thermi phylum and Bacteroides, Candidatus Cardinium, and Leptospirillum genera Phytogenics Essential oils 100 ppm 9 weeks 25% thymol and 25% carvacrol Gilthead seabream ↓ FGR ↑ Intestine mucosal foldings Induce an anti-inflammatory and anti- Perez-Sanchez, combination (commercial additive) (Sparus aurata) ↑ Intestine enterocytes proliferative transcriptomic profile with Benedito-Palos ↑ Intestine goblet cells probable improvement in the (108) ↓ Expression of genes related to cell absorptive capacity of the intestine differentiation and proliferation, intestinal architecture and June 2021 | Volume 12 | Article 695973 permeability, immunosurveillance, Phytogenics and Fish Mucosal Immunity such as cytokines, in the intestine SGR, Specific Growth Rate. FCR, Feed Conversion Ratio. FGR, Feed Gain Ratio. OTU, Operational Taxonomic Unit. *Indicate the putative plant species with high content of the referred bioactive compound. N/I, not identified or not assessed. Studies reporting the application of plant extracts or related compounds as bath treatments or evaluating bactericidal or antiparasitic effects in vitro were excluded from the selection. The table omits systemic immunity-related results, digestive enzymes or other complementary analysis performed within each study. Blends with other components besides the selected group of plants, terpenes or organosulfur compounds were excluded as well. Firmino et al. Phytogenics and Fish Mucosal Immunity mucus, few studies have tried to explain and characterize the conditions and acquire relevant data for their further immunomodulatory mechanisms underlying such responses development and general and safe use. neither which specific compounds might be exerting such effects. The effect of some single specific bioactive compounds related Carvacrol, thymol, p-cymene and g‐terpinene are identified as to aromatic plants of the Lamiaceae family, such thymol or the predominant bioactive compounds of most of the members carvacrol, upon mucosal tissues were also reported (Table 1). of the Lamiaceae family considered in this review, which were Although these studies have the advantage to associate a specific mainly found in oregano and thyme. In addition, peppermint, mucosal response to the administration of a specific compound, rosemary and basil contain preponderant concentration of other once again it is observed that most of them only reported the bioactive compounds such menthol, eugenol and 1,8-cineole analysis of few immune and oxidative markers (97, 98, 101, 103), (Table 1). Interestingly, some studies have reported carvacrol or a limited microbiological examination (99, 100). In fact, few and 1,8-cineole presence in the fish skin mucus (63, 81). This studies were observed to apply complementary analysis, or phenomenon of bioactive compounds efflux through skin could achieved to successfully discuss the multifactorial impact be responsible for the immunomodulatory and antimicrobial exerted by such nutritional strategies upon mucosal tissues effects observed in the fish skin mucus. However, most of the (107, 108). reviewed studies did not report the phytogenics composition Although the overall data suggest the therapeutic potential of neither the assessment of their translocation through mucus. phytogenics derived from Lamiaceae family of plants in Some studies have also reported a protective effect through aquafeeds, especially of their associated terpene phenolic the reduction in gills’ histopathological lesions induced by toxic compounds, unfortunately none of the studies has proposed element exposure or pathogenic challenges (62, 66, 85). accurate mechanisms that could be responsible for the broad Contrarily to the studies describing the effects of phytogenics effects of these metabolites described upon fish mucosal tissues. upon skin mucus secretion and their immunomodulatory Despite the lack of reliable information for aquaculture relevant potential, their impact upon the GIALT is very scarce, being fish species, it is possible that the above-mentioned antimicrobial mostly limited to histological observations. Similarly, studies on properties of these compounds, their free radicals’ scavenging the impact of phytogenics upon the intestine are commonly ability, along with their aptitude to activate TRP channels that focused on evaluating alterations in morphoanatomical modulate inflammatory processes may underlie the parameters such as an increase in villus length, width and immunomodulatory properties and microbiota modulation goblet cells count, which are usually associated to described in different mucosal tissues. improvements in fish growth performance (59, 60, 64, 84, 85). Some studies have also reported the modulation in the number of Effect of Dietary Organosulfur Compounds intestinal lymphocytes (70, 72). Other authors have described a Upon Fish Mucosal Immunity positive impact upon the gastrointestinal activity of humoral The main constituents of extracts and essential oils from Allium sp., immune markers, such lysozyme (86), the activity of antioxidant such as garlic (Allium sativum, Alliaceae) and onion (Allium cepa, enzymes and oxidative markers (67, 106) or the down-regulation Alliaceae), are sulfur‐containing compounds. This group of of the expression of pro-inflammatory genes, such tnfa and tnfb bioactive substances comprises alliin, allicin and its derived (61). Besides, the beneficial impact of phytogenics administration bioactive compounds like ajoene, diallyl trisulfide (DATS), diallyl upon the intestine microbiota composition was also suggested disulfide (DADS), diallyl sulfide (DAS) and allyl methyl disulfide, (61, 87). Nonetheless, analysis described were constantly commonly termed as organosulfur compounds. These organosulfur incomplete in terms of mucosal immune response evaluation, compounds are the responsible for the recognized antioxidative, since limited classical immune or oxidative markers were antimicrobial, antifungal and antiparasitic properties of garlic (126). assessed in each of the above-mentioned studies. Similarly, Allicin (S-allyl-2-propenyl thiosulfinate) is usually the main microbiota studies were often restricted to a particular group biologically active component of garlic and related species; of bacteria, such lactic bacteria, failing to properly characterize however, it is highly unstable under physiological conditions; microbiota functionality and modulation by the experimental thus, quickly being transformed into its organosulfur derivates, diets; thus, resulting in partial and biased conclusions when which also exhibit therapeutic properties (127–129). assessing the regulatory effects of functional feed additives on Organosulfur compounds have been particularly studied for mucosal tissues. their antiparasitic activity. Among them, ajoene was described to In addition, some studies reported no effect of the administrated interfere with parasite and host cell membrane protein and lipid phytogenics upon mucosal parameters (68, 69, 80, 82, 83). Such trafficking, with irreversible detrimental consequences for the discrepancies among studies evaluating a particular plant extracts parasite (130). This is of special relevance since teleost mucosal may be due to the diversity of the referred studies in terms of tissues are known to have a high constitutive expression of Th2 experimental design, fish species selected, plant origin, markers that indicate a skewed immune response targeted supplemented form and inclusion level of phytogenetics, among against parasites (131). Regarding their bactericidal properties, others. This miscellaneous of studies and the lack of protocols the organosulfur compounds can penetrate the bacterial cell allowing appropriate additive and animal testing, highlights the membranes, cause changes in the structure of thiol (-SH) urgent need to standardize the experimental designs and procedures containing enzymes and proteins, and lower the expression of in order to properly evaluate these compounds under in vivo important genes involved in the QS in bacteria, inhibiting the Frontiers in Immunology | www.frontiersin.org 11 June 2021 | Volume 12 | Article 695973 Firmino et al. Phytogenics and Fish Mucosal Immunity growth of both Gram-positive and Gram-negative bacteria (132). inflammatory and oxidative markers in gills (89, 96) and The higher the number of sulfur atoms present in the intestine (90, 94–96, 102), or incomplete microbiological compounds, the more is its bactericidal activity (133). examination that lacks in-depth the functional interpretation Therefore, the administration of organosulfur-containing of their mode of action at cellular level (87, 90, 92). phytogenics may induce important changes in the fish Overall, studies reporting the effect of the administration of mucosal-associated microbiota with potential effects upon the phytogenics derived from Allium sp. upon fish mucosal tissue mucosal immunity. suggest the health-promoting potential of the organosulfur The detoxification and chemoprotective benefits from various compounds that characterize this group of plants. However, organosulfur compounds have been associated to their ability to there is currently no robust studies under a pharmacological scavenge free radicals and selectively enhance or suppress the point of view that intent to demystify the accurate mechanisms levels genes or proteins of several antioxidant enzymes, such as responsible for the effects described on fish mucosal tissues, cytochrome P450 enzymes or glutathione S-transferase (GST) whose lack of reliable information critically restrains their (134), exerting a direct effect upon immune cells (135). In this application as potential functional feed additives in aquafeeds. line, their anti-inflammatory activity upon immune and Similar to the terpene phenolic compounds, the organosulfur intestinal epithelial cells was associated to the inhibition of compounds have also recognized antipathogenic and antioxidant ROS production and the modulation of the NF-kB and MAPK properties, in addition to their common ability to activate TRP signaling pathways (136, 137). Some organosulfur compounds, channels that modulate inflammatory processes. Since in higher such allyl sulphides, were also observed to increase the levels of vertebrates dermal emission of organosulfur compounds were anti‐inflammatory H2S in intestinal epithelial cells, promoting demonstrated after garlic ingestion (147). In this sense, the efflux mucosal integrity, tissue repair and stimuli perception (138). In of organosulfur compounds through the integument could be accordance, organosulfur compounds, such as DADS, are also also playing a critical role in the recurrently reported effects of donors of H2S, whose positive effects upon the intestinal health dietary garlic and other aromatic plants in fish mucus, as could be also produced through the modulation of the enteric previously referred for carvacrol and 1,8-cineole. microbiota (139). In addition, organosulfur compounds were observed to promote mucin expression in human airway Effect of Combinations of Phytogenics epithelial cells, being suggested to improve the mucosal Derived From Lamiaceae and Allium sp. epithelial barrier function (140, 141). Upon Fish Mucosal Immunity Conversely, these organosulfur compounds have been also Some studies have reported the beneficial effects of the reported to stimulate inflammatory immune responses, combination of Lamiaceae and Allium sp. phytogenics upon promoting the release of pro-inflammatory cytokines, enhancing fish mucosal tissues (Table 1). For instance, in European seabass the proliferation of lymphocytes, macrophage phagocytosis and (Dicentrarchus labrax) a combination of garlic and essential oils modulating the infiltration of immune cells (142). For instance, it from plants of the Lamiaceae family promoted skin mucus was demonstrated that some allyl-containing organosulfur lysozyme activity and fish survival against V. anguillarum compounds directly activate Ca2+ flux in neutrophils augmenting when exposed to a confinement stress (104). In greater their phagocytic function and consequent ROS production. In amberjack (Seriola dumerili), the same additive induced an up- parallel, other compounds are able to inhibit spontaneous ROS regulation of a set of immune related genes in the skin in production by neutrophils (143). This apparent antagonistic effect response to a monogenean parasite Neobenedenia girellae evidences the pleiotropic protective effects of garlic extracts and infection (16). In accordance, a blend of garlic essential oils, essential oils, being simultaneously capable of inducing immune carvacrol and thymol was also reported to positively impact both responses and anti-inflammatory counteractions. Moreover, as gilthead seabream (Sparus aurata) skin mucus in terms of previously suggested for the mode of action of terpene phenolic bacterial inhibition capacity against fish pathogens and compounds, organosulfur compounds are also able to activate TRP decrease of stress markers, whereas the transcriptional analysis channels, TRPA1 and TRPV1 channels in particular (144–146), suggested the stimulation of the secretory pathway possibly suggesting the Ca2+ induced cellular immune activation (143). associated to humoral immune molecules secretion into mucus Garlic has been for long studied and recognized for its and activation of phagocytic cells (14). The same blend was benefits as growth- and flesh quality-promoting effects in reported to regulate the transcription of genes related to immune cultured fish, as well as for its antibacterial and antiparasitic response in gills, which was mediated by granulocytes, as well as properties (51). However, there is scarce information regarding sustaining both anti-inflammatory and antioxidative responses. the activity of garlic-derived organosulfur bioactive compounds In addition, the above-mentioned study revealed that the tested upon fish mucosal tissues. The synthesis of the results from phytogenic compounds promoted the presence of sialic-acids several studies reporting the health promoting properties of containing glycoproteins in both epithelial and mucous cells, phytogenics derived from Allium sp. are shown in Table 1. which globally resulted in a decrease in the intensity of gills’ Similar to studies testing phytogenics from Lamiaceae family infestation by the monogenean ectoparasite Sparicotyle origin, the evaluation of the supplementation of phytogenics chrysophrii in gilthead seabream (15). Both, referred derived from Allium sp., also focuses on few immune markers in phytogenics combinations were observed to positively affect skin mucus (88, 93), histopathological observations or some the gut health status of those fish species by improving the Frontiers in Immunology | www.frontiersin.org 12 June 2021 | Volume 12 | Article 695973 Firmino et al. Phytogenics and Fish Mucosal Immunity protective intestine mucus coverage post-challenge (13), level of the main mucosal lymphoid tissues in fish is depicted regulating the intestine immune transcription (12) and in Figure 1. modulating their intestinal microbiota (12, 105). In addition, a summarized representation of the potential According to the above-described studies and the acknowledged mucosal immune responses induced by the dietary administration properties of these phytogenics’ bioactive compounds, we suggest of terpene and/or organosulfur based phytogenics and their effects that the mechanisms of cell activation that may be responsible for against fish pathogenic organisms is suggested in Figure 2. In this the mucosal immune-related responses are mediated by the representation, the holistic perspective of the compounds’ effects activation of TRP cation channels in both immune and epithelial upon the most studied mucosal-associated lymphoid tissues in fish cells of mucosal tissues. The bioactive compounds may activate TRP so far – gill, gut, and skin – as targets for oral immunostimulation is channels leading to intracellular Ca2+ increase and the activation of highlighted through the stimulation of both humoral and cellular the TAK1/MAPK/NF-kB signaling pathways, modulating the immunity, mucosal secretion, microbiota modulation and other expression of pro-and anti-inflammatory cytokines, and potential physiological and metabolic responses. antioxidative enzymes such as cytochrome P450. In parallel, stimulation by pathogen-associated molecular patterns (PAMPs), who might be also modulated by the antimicrobial properties of FUTURE PERSPECTIVES these compounds, may facilitate the activation of TLR and TRP signaling pathways; thus, amplifying the mucosal immune Feeding the projected world population by 2050 in a sustainable responses. Moreover, the bioactive compounds are also suggested way is a great challenge, in which aquaculture is predicted to supply to passively diffuse across the cell membrane, scavenging ROS that the majority of aquatic dietary protein. For that, the implementation contribute to the inflammatory pathways, and interacting with TRP of novel policies and production system approaches targeting channels of the endoplasmic reticulum, potentially stimulating the effective health management and animal welfare are mandatory secretory pathway. The above-described mode of action of (1). Moreover, indiscriminate prophylactic use of antibiotics phytogenics derived from Lamiaceae family and Allium sp. at the associated to intensive aquaculture practices can still be observed FIGURE 1 | Suggested mechanisms of cell activation by the transient receptor potential (TRP) cation channels mediated by phytogenics’ bioactive compounds in mucosal-associated lymphoid tissues (MALTs). Bioactive compounds activate TRP channels leading to intracellular Ca2+ increase and non-canonical activation of the TAK complex. In parallel, stimulation by pathogen-associated molecular patterns (PAMPs) may facilitate the activation of TLR and TRP signaling pathways. Modified from Galindo-Villegas, et al. (124). TLR, toll-like receptors; MyD88, myeloid differentiation primary response 88; TAK, transforming growth factor beta (TGFb) activated kinase; TAB, TGFb activated kinase binding protein; ROS, reactive oxygen species; NF-kB, nuclear factor kappa-B; IKK, inhibitor of NF-kB kinase; MAPK, mitogen- activated protein kinase; AP1, activator protein 1; CYP, cytochromes P450; P, phosphorylation. Frontiers in Immunology | www.frontiersin.org 13 June 2021 | Volume 12 | Article 695973 Firmino et al. Phytogenics and Fish Mucosal Immunity FIGURE 2 | Summary of the proposed mode of response induced by phytogenic bioactive compounds in fish mucosal tissues. The effects against representative types of common pathogens on the most studied mucosal-associated lymphoid tissues in fish so far are represented in the figure. (A) Gills. (B) Gut. (C) Skin. Gilthead seabream (S. aurata) was used in the figure as a representative aquaculture-relevant fish. among some of the major aquaculture producing countries, as it has forms in different fish species that are generally randomly selected. In been recently reviewed (148, 149). However, in 2022, several addition, a considerable number of these studies provide little or countries, including the EU will prohibit all forms of routine partial information regarding the effect of a given immunostimulant, antibiotic use in farming, including preventative group treatments since their definitive efficiency assessment rely on the evaluation of which highlights the necessity for the development of more basic biochemical parameters that are to some extent obsolete if sustainable alternative treatments (150). Under this context, the compared against the actual state-of-the-art. Based on the former market for sustainable products and feed additives is increasingly idea, diverse omics tools available play a fundamental role for proper growing and the number of studies on the use of a wide variety understanding and characterization of their mode of action in phytogenics as sustainable tools to be implemented in aquaculture mucosal tissues at cellular level. Together, these factors question the production has increased dramatically in the last decade (5). The reproducibility and safety of a large number of studies available and global market of phytogenic feed additives including major tier I limits the use of several of those proposed substances in commercial and II suppliers was estimated on 753M USD in 2020 and it is functional feeds. projected to reach 1,098M USD by 2025 (151). In this regard, we propose that the study of purified bioactive Although limitations in testing and reproducing studies using compounds may represent a viable solution to circumvent dietary immunostimulants have been pointed out since long ago variability, and the biological mode of action of isolated (152–155), the current knowledge on the pathways and mechanisms compounds should be primarily assessed in vitro under variated followed by these compounds at the cellular level is still neglected. A settings, before moving forward to in vivo trials. However, it is large selection of experimental designs, fish species, phytogenics tested important to consider that the biological activities of essential oils or and diet composition fails in the association of selected bioactive extracts cannot be attributed to a single compound or to a unique compounds to specific effects. Moreover, it is important to consider specific mechanism, since their multi-component properties exert the difficulties to carry out comparison among the available studies greater biological activity when compared to the major components because of the different assays, testing methods, different extraction alone, whose function is probably regulated by the synergy with procedures of plant essential oils or extracts, and the intrinsic limiting compounds (8). In this light, essential oils and extracts from variation in chemical phytoconstituents in plants due to different different plants have been explored for their potential as resistance agroclimatic conditions, harvesting season and plant phenotype. This modifying agents (156). While their chemical complexity may essential oils or extracts consist of a variable mixture of different represent a clear advantage in terms of reducing the risk of bioactive molecules that area generally not characterized, and are inducing bacterial adaptation and resistance to single compounds, administrated through variable periods of time, dietary doses and or even promoting a wider antibacterial activity, the use of blends of Frontiers in Immunology | www.frontiersin.org 14 June 2021 | Volume 12 | Article 695973 Firmino et al. Phytogenics and Fish Mucosal Immunity phytogenics makes difficult to proper characterize their mode of storage or along the gastrointestinal tract. The overall limitations action. Therefore, when developing such additives, the formulation identified in most of the currently available studies assessing fish of combined purified compounds through the correct and soundly immunity leads to the stigmatization of phytogenics application, in in vitro functional characterization to obtain potential synergies is which compounds with high pharmacological value are labeled under recommended. Moreover, long-term studies assessing whether the the “medicinal plants” or “herbalism” pseudoscience stigma, with bioactive compounds, single or combined, induce bacterial disbelieving scientific evidence. This represents a major restriction for tolerance, transmissible adapted resistance or any other change on the development of effective phytogenics at commercial scale. a large scale should be implemented and the effects on both beneficial and pathogenic bacteria determined through in vitro, in silico and finally in vivo approaches (103, 157). CONCLUSIONS While several phytogenics have been proved remarkably Overall, it is fundamental that the efforts made in the research for efficient in promoting mucosal fitness (9), little efforts have sustainable prophylactic tools to boost host’s immune condition, been made to elucidate the underlying pharmacokinetics and stress resistance and pathogenesis prevention will culminate on immunostimulatory mechanisms of tested compounds upon the reliable administration strategies for the aquaculture sector. MALTs, with few in vitro studies published to date. It should also Among the most studied group of natural bioactive be highlighted that occasionally in vitro studies do not accurately compounds, both terpenes and organosulfur compounds have translate into predictable responses in vivo (83); thus, both in been suggested to display antimicrobial, antioxidant, anti- vitro and in vivo studies should be performed whenever possible. inflammatory and immunomodulating activities, with the This lack of complementary information supports the demand potential of improving fish mucosal barrier function and for additional profound research on the fate and length in which integrity. Although they comprise a promising group of particular phytogenic compounds act, which is crucial for further phytogenics for aquafeeds, an urgent update in the academical developing functional additives and their application in an approach and experimental methodologies are needed to industrial context. Although the specific mechanisms behind elucidate their pharmacokinetics and mode of action in depth. the observed fish mucosal physiological responses are still poorly Therefore, in the present review we propose important molecular described, it is possible that cellular pathways involving the signaling pathways and hypothesize their involvement on the activation of TRP receptors by the bioactive compounds might dietary immunomodulation in fish by the selected phytogenics. be responsible for the reported mucosal immune responses. Besides, this response might be potentiated by the PAMP- induced activation of the TLR cell-signaling cascade, as AUTHOR CONTRIBUTIONS synthetized in Figure 1, which would explain the fish improved ability to cope with pathogenic challenges. Thus, it is JF and JG-V data analysis and visualization. JF wrote the draft. advisable in nutritional dose-response evaluating phytogenics to All authors contributed to the article and approved the evaluate changes in expression in TRP receptors as well as gene submitted version. markers of the TAK1/MAPK/NF-kB signaling pathways in order to provide insight into their mode of action at mucosal level. ACKNOWLEDGMENTS Another limitation that should be taken into account when testing phytogenics is that most plant-derived bioactive compounds are Authors thank the support of the projects: (1) Nutritional either volatile and/or susceptible to rapid degradation in the stomach strategies for the improvement of productive performance: the where acid digestion takes place, with consequent low availability at use of functional feeds and health diets in aquaculture the intestinal level or uncontrolled changes in the dose of (DIETAplus), funded by JACUMAR (Ministry of Agriculture, administration. Hence, to overcome this limitation and minimize Fisheries and Environment of Spain, MAPAMA) and FEMP (EU); potential losses, controlled releasing techniques, such as (2) LARVAplus network Strategies for the development and encapsulation or other coating technologies, can be used to improvement of fish larvae production in Ibero-America improve the proper delivery of phytogenics. This technology allows (117RT0521) funded by the Ibero-American Program of Science a prolonged absorption and local availability of the bioactive and Technology for Development (CYTED, Spain); (3) FR-L compounds along the gastrointestinal tract, ultimately increasing thanks the support of Fondecyt regular grant (project number their beneficial impact upon the host (158, 159). Moreover, 1211841; Agencia Nacional de Investigació n y Desarrollo [ANID]; encapsulation protects phytogenics from environmental Government of Chile); (4) JF has been subsidized by the Industrial degradation, such as from light, temperature and/or pH variations, PhD program of Generalitat de Catalunya and TECNOVIT- and eventually playing an important role in their palatability, FARMFAES S.L. (Nb. 2017 DI 017). The funder was not masking the potential pungency associated to some compounds involved in the study design, collection, analysis, interpretation that otherwise can affect feed intake (158). It is important to of data, the writing of this article or the decision to submit it for highlight that most of the studies considered in this review did not publication. (5) European Research Council (ERC) under the take into consideration those aspects, administrating phytogenics as European Union’s Horizon 2020 research and innovation powder forms, hydroethanolic extracts or dissolved solutions without programme (grant 683210). Nord University Access Fund proper assessment of their potential biodegradation during feed covers the OA publication cost. Frontiers in Immunology | www.frontiersin.org 15 June 2021 | Volume 12 | Article 695973 Firmino et al. Phytogenics and Fish Mucosal Immunity REFERENCES 16. Ferná ndez-Montero Á ., Torrecillas S, Acosta F, Kalinowski T, Bravo J, Sweetman J, et al. Improving Greater Amberjack (Seriola dumerili) 1. Costello C, Cao L, Gelcich S, Cisneros-Mata >M.Á ., Free CM, Froehlich HE, Defenses Against Monogenean Parasite Neobenedenia Girellae Infection et al. The Future of Food From The Sea. Nature (2020) 588(7836):95–100. Through Functional Dietary Additives. 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Measurement of Diallyl Disulfide any commercial or financial relationships that could be construed as a potential and Allyl Methyl Sulfide Emanating From Human Skin Surface AND conflict of interest. Influence of Ingestion of Grilled Garlic. Sci Rep (2020) 10(1):465. doi: 10.1038/s41598-019-57258-1 Copyright © 2021 Firmino, Galindo-Villegas, Reyes-López and Gisbert. This is an 148. Lulijwa R, Rupia EJ, Alfaro AC. Antibiotic use in Aquaculture, Policies and open-access article distributed under the terms of the Creative Commons Attribution Regulation, Health and Environmental Risks: A Review of the Top 15 Major License (CC BY). The use, distribution or reproduction in other forums is permitted, Producers. Rev Aquaculture (2020) 12(2):640–63. doi: 10.1111/raq.12344 provided the original author(s) and the copyright owner(s) are credited and that the 149. Schar D, Klein EY, Laxminarayan R, Gilbert M, Van Boeckel TP. Global original publication in this journal is cited, in accordance with accepted academic Trends in Antimicrobial Use in Aquaculture. Sci Rep (2020) 10(1):21878. practice. No use, distribution or reproduction is permitted which does not comply with doi: 10.1038/s41598-020-78849-3 these terms. Frontiers in Immunology | www.frontiersin.org 20 June 2021 | Volume 12 | Article 695973