Phytogenic Bioactive Compounds Shape Fish Mucosal Immunity

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Phytogenic Bioactive Compounds Shape Fish Mucosal Immunity
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
Phytogenic Bioactive Compounds Shape Fish Mucosal Immunity
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,
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                                                                                                                                                                                                    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)
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                                                                                                                                                                                                                                                                                                                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
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                                                                                                                                                                                                    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

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