Phytogenic Bioactive Compounds Shape Fish Mucosal Immunity
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REVIEW
published: 18 June 2021
doi: 10.3389/fimmu.2021.695973
Phytogenic Bioactive Compounds
Shape Fish Mucosal Immunity
Joana P. Firmino 1,2,3, Jorge Galindo-Villegas 4*, Felipe E. Reyes-López 5,6,7
and Enric Gisbert 1
1 Aquaculture Program, Institut de Recerca i Tecnologia Agroalimentàries (IRTA) Centre de Sant Carles de la Ràpita (IRTA-
SCR), Sant Carles de la Ràpita, Spain, 2 PhD Program in Aquaculture, Universitat Autònoma de Barcelona, Bellaterra, Spain,
3 R&D Technical Department, TECNOVIT – FARMFAES, S.L., Alforja, Spain, 4 Faculty of Biosciences and Aquaculture, Nord
University, Bodø, Norway, 5 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona,
Bellaterra, Spain, 6 Facultad de Medicina Veterinaria y Agronomı´a, Universidad de Las Américas, Santiago, Chile, 7 Consorcio
Tecnológico de Sanidad Acuı´cola, Ictio Biotechnologies S.A., Santiago, Chile
Aquaculture growth will unavoidably involve the implementation of innovative and
Edited by:
sustainable production strategies, being functional feeds among the most promising
Javier Santander, ones. A wide spectrum of phytogenics, particularly those containing terpenes and
Memorial University of Newfoundland,
organosulfur compounds, are increasingly studied in aquafeeds, due to their growth
Canada
promoting, antimicrobial, immunostimulant, antioxidant, anti-inflammatory and sedative
Reviewed by:
Seyed Hossein Hoseinifar, properties. This trend relies on the importance of the mucosal barrier in the fish defense.
Gorgan University of Agricultural Establishing the phytogenics’ mode of action in mucosal tissues is of importance for
Sciences and Natural Resources, Iran
Débora Torrealba,
further use and safe administration. Although the impact of phytogenics upon fish
Pontificia Universidad Católica de mucosal immunity has been extensively approached, most of the studies fail in
Valparaı´so, Chile
addressing the mechanisms underlying their pharmacological effects. Unstandardized
*Correspondence:
testing as an extended practice also questions the reproducibility and safety of such
Jorge Galindo-Villegas
jorge.galindo-villegas@nord.no studies, limiting the use of phytogenics at commercial scale. The information presented
herein provides insight on the fish mucosal immune responses to phytogenics, suggesting
Specialty section: their mode of action, and ultimately encouraging the practice of reliable and reproducible
This article was submitted to
Comparative Immunology, research for novel feed additives for aquafeeds. For proper screening, characterization
a section of the journal and optimization of their mode of action, we encourage the evaluation of purified
Frontiers in Immunology
compounds using in vitro systems before moving forward to in vivo trials. The
Received: 15 April 2021
Accepted: 31 May 2021
formulation of additives with combinations of compounds previously characterized is
Published: 18 June 2021 recommended to avoid bacterial resistance. To improve the delivery of phytogenics and
Citation: overcome limitations associated to compounds volatility and susceptibility to degradation,
Firmino JP, Galindo-Villegas J, the use of encapsulation is advisable. Besides, newer approaches and dedicated
Reyes-López FE and Gisbert E (2021)
Phytogenic Bioactive Compounds methodologies are needed to elucidate the phytogenics pharmacokinetics and mode of
Shape Fish Mucosal Immunity. action in depth.
Front. Immunol. 12:695973.
doi: 10.3389/fimmu.2021.695973 Keywords: immunity, MALT, organosulfurs, terpenes, sustainable aquaculture, teleost, phytogenic additive, TRPV4
Frontiers in Immunology | www.frontiersin.org 1 June 2021 | Volume 12 | Article 695973
Firmino et al. Phytogenics and Fish Mucosal Immunity
INTRODUCTION (12–16). Besides, the immune system influences the regulation
and composition of the microbiota and vice versa, an interaction
Sustainable food supply to feed the demand of the projected world that plays a determinant role in the maintenance of the mucosal
population by 2050 is a challenge, in which aquaculture is predicted integrity and functionality (17, 18). Hence, both the improvement
to be the main source of aquatic dietary proteins. Such growth will of the mucosal barrier characteristics and the modulation of the
unavoidably involve the implementation of innovative aquaculture microbiota are central targets for the development of new
production strategies, targeting issues related to effective health phytogenic additives, while understanding their mode of action
management and animal welfare (1). In this regard, the at cellular and molecular levels is critical for elucidating their
development and application of functional feeds represent a benefits to the host.
sound strategy to improve aquaculture systems, since they provide Given the extended literature available on plant-based
functional health benefits to animals beyond their nutritional value functional additives and the significance of the mucosal
(2). In this scenario, phytogenics, also known as phytobiotics, are immunity described above, our review efforts focus exclusively
defined as environmentally friendly plant-derived bioactive on the physiological and immunological responses achieved by the
compounds used as functional feed additives that show positive most studied fish mucosal tissues, intestine, gills and skin, of
effects on animal growth and health. Phytogenic often comprise organisms fed with phytogenics of the Lamiaceae family and
aromatic plants extracts, and essential oils characterized by its Allium sp. In the first part, we present a thorough description
richness in biologically active compounds (3, 4). In farmed fish, a on their main bioactive compounds and relevant biological
wide spectrum of phytogenics have been increasingly studied properties. Then, the immunomodulatory properties and the
mainly due to their wide repertoire of properties, including mechanisms they can trigger on the fish mucosal tissues are
growth promotion, and antimicrobial, immunostimulant, explored and further potential mechanisms hypothesized.
antioxidant, anti-inflammatory and sedative activities (5). In Finally, research gaps and constrains for the development of
particular, phytogenics derived from Lamiaceae family and Allium applicable phytogenic-based additives are discussed. Overall, the
sp. are among the most commonly studied and administrated plant- information presented herein aims to provide clear insights on the
based additives (6, 7). Nonetheless, the complexity of the fish mucosal immune response dietary treated with phytogenics,
mechanisms of action and the pharmacological effects promoted propose viable mechanisms for exploiting them, and ultimately
by the diverse bioactive compounds present in such plants, along encourage the practice of reliable and reproducible research for the
with their frequently observed synergistic behavior (8), often limits development of novel feed additives to be used as sustainable and
the full understanding of their biological activity (9). safe prophylactic strategies for aquaculture.
Since outbreaks of fish diseases are one of the main constrains
for the progress of the aquaculture sector (10), the inclusion of
phytogenics’ in aquafeeds is achieving significant attention at a FISH MUCOSAL IMMUNITY AT A GLANCE
global scale. The impact of phytogenics upon fish systemic
immunity has been extensively tested in the past (5, 9). The mucosal barrier constitutes the fish first line of defense
However, an increasing trend to evaluate phytogenics’ impact against the surrounding environment and potential pathogens.
upon the mucosal immunity has been gained importance in Fish mucosal tissues are particularly characterized by a mucosa-
recent years, which is mainly attributed to the importance of the associated lymphoid tissue (MALT), harboring diverse myeloid
mucosal barrier in the fish defense against variated threats and, and lymphoid cells that are responsible for the host protection
potential colonization and invasion by pathogenic organisms against pathogens and antigens, while tolerating beneficial
(11). In contrast, most of the studies evaluating the effect of symbiont colonization to maintain mucosa homeostasis (19–
phytogenics in fish systemic immunity are only supported by a 21). Six different MALTs have been described so far in teleosts.
selection of repetitive primary analyses (i.e., lysozyme, The gut-associated lymphoid tissue (GALT), the gill-associated
bacteriolytic and complement activities, immunoglobulins, lymphoid tissue (GIALT), the skin-associated lymphoid tissue
oxidative stress enzymes, etc.) serving only as proxies, that (SALT), the nasopharynx-associated lymphoid tissue (NALT)
only provide a snapshot of the effects of the evaluated feed and, the more recently characterized the buccal, and pharyngeal
additive on the organism. These approaches do not allow MALTs (22). Other mucosal immune systems have been
elucidating their mode of action at cellular and molecular hypothesized and are currently under study (23). Despite the
levels. This is of special relevance when dealing with functional existence of others, the GALT, GIALT and SALT are the most
feed additives with potential pharmacological properties, as their studied and well characterized MALTs and therefore selected as
standardized use mainly depends on the proper understanding of target in this review.
their regulatory properties in the immune system. Among the extensive cell types with immune capacity
Phytogenic administration has the potential to regulate the coexisting in the fish body, upon sensing the presence of
mucosal barrier function by means of several molecular pathogenic or commensal microbe-associated molecular
mechanisms, in which the phytogenic bioactive compounds pattern (MAMP) a downstream signaling response mediated
interact with cellular transcription factors and metabolic through pattern recognition receptors (PRRs) immediately takes
cascades. Therefore, the modulation of the expression of genes place. So far, several piscine PRRs have been identified, being the
coding for immune relevant molecules alter the mucosal toll-like receptors (TLR), NOD-like receptors (NLR) and retinoic
protective characteristics and their immunological status acid-inducible gene I (RIG-I)-like receptors (RLRs) the best
Frontiers in Immunology | www.frontiersin.org 2 June 2021 | Volume 12 | Article 695973Firmino 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 695973Firmino 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 695973TABLE 1 | Extended summary of the current available literature on nutritional effects of Lamiaceae family and Allium sp. derived phytogenics upon fish mucosal immune response.
Frontiers in Immunology | www.frontiersin.org
Firmino et al.
Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference
plant origin form dosage(s) administration components (≤ 3)
tested
Lamiaceae family
Oregano Powder 0.5%, 8 weeks N/I Zebrafish (Danio ↑ Final weight ↑ Skin mucus lysozyme activity Beneficially affects the skin mucus Rashidian, Boldaji
(Origanum 1.0% and rerio) ↑ Weight gain ↑ Skin mucus alkaline phosphatase immune parameters, growth (56)
vulgare) 2.0% ↓ FCR activity performance and survival against
↑ SGR ↑ Skin mucus total Ig pathogenic bacterial challenge
↑ Survival ↑ Skin mucus protease activity
against A. ↑ Skin mucus total protein
hydrophila
Oregano Powder 0.5% and 15 and N/I Gilthead seabream No effect ↑ Skin mucus IgM Oregano improves humoral immunity Beltrá n, Gonzalez
(Origanum 1.0% 30 days (Sparus aurata) ↑ Skin mucus bactericidal activity and increases the antibacterial activity Silvera (57)
vulgare) against P. damselae of skin mucus
Oregano Ethanolic extract 0.2% and 60 days + 7 N/I Nile tilapia ↑ Final weight ↑ Skin mucus total Ig Can effectively improve the fish Mohammadi,
(Origanum 0.5% days A. (Oreochromis ↑ Weight gain growth, health, and immune status Rafiee (58)
vulgare) hydrophila niloticus) ↓ FCR
challenge ↑ SGR
↑ Survival rate
↑ Survival
against A.
hydrophila
Oregano Powder 5.0, 10.0, 15.0 8 weeks Carvacrol and thymol Common carp ↑ Final weight ↑ Intestine villus height Dose-dependent enhancement of Abdel-Latif, Abdel-
(Origanum and 20.0 g kg- (Commercial product) (Cyprinus carpio) ↑ Weight gain ↑ Intestine villus width intestinal morphometry, which Tawwab (59)
vulgare) 1
↑ SGR ↑ Intestine crypt depth subsequently lead to improvement of
nutrients absorption
Oregano Essential oils 0.75, 1.5, 2.25 64 days Carvacrol, thymol and Nile tilapia No effect ↑ Intestine villus height Increases intestinal villus size Heluy, Ramos (60)
(Origanum and 3.0 g kg-1 p-cymene (Oreochromis
vulgare) niloticus)
Oregano Essential oils 0.5, 1.5 and 8 weeks + 7 N/I Koi carp (Cyprinus ↑ Survival ↓ TNF-a and TGF-b gene expression Presents immunomodulatory effects Zhang, Wang (61)
(Origanum 4.5 g kg-1 days A. carpio) against A. in intestine and enhances disease resistance.
vulgare) hydrophila hydrophila ↑ Actinobacteria phylum, and Also beneficially alters the gut bacterial
challenge Propionibacterium, Brevinema and community composition of fish
5
Corynebacterium genera
↓ Bacteroidetes phylum and Vibrio
genus
No effect on bacterial alpha diversity
Oregano Powder 0.5% and 30 days + Carvacrol and thymol Common carp N/I ↓ Gill histopathologic lesions Protective roles against the adverse Khafaga, Naiel (62)
(Origanum 1.0% cypermethrin (Commercial product) (Cyprinus carpio) ↓ Gill proliferating cell nuclear antigen effects of cypermethrin, enhancing
vulgare) exposure (PCNA) and caspase-3 immune recovery from the exposure
positive cells
Oregano Essential oils 0.01%, 0.02%, 24 days + 28 Carvacrol, p‐cymene and g‐ Chum salmon ↑ feed efficiency Carvacrol content detected in the skin Preventive effects against I. salmonis Mizuno, Urawa
(Origanum 0.05% and days I. salmonis terpinene (Oncorhynchus keta) ↓ I. salmonis of fish fed the oregano supplemented and T. truttae and suggests the (63)
vulgare) 0.10% and T. truttae infection diet possibility that its anti‐parasitic action
challenge ↓ T. truttae is attributable to the bioactive
(Total 52 days) infection component emergence through the
↓ cumulative skin
mortality
Oregano Essential oils 0.5, 1.0, 1.5, 90 days N/I Yellow N/I ↑ Intestine villus length Promotes increased absorption Ferreira, Caldas
(Origanum 2.0 (Commercial product) tail tetra (Astyanax ↑ Intestine villus width surface area and modulates the (64)
vulgare) and 2.5 g kg-1 altiparanae) ↑ Intestine absorption area number of goblet cells involved in
↑↓ Intestine goblet cells number
June 2021 | Volume 12 | Article 695973
protecting the intestinal mucosa
Phytogenics and Fish Mucosal Immunity
Marjoram Ethanolic extract 0.1, 0.2 and 60 days + 10 N/I Common carp ↑ Final weight ↑ Skin mucus alkaline phosphatase Increase fish skin mucosal immunity Yousefi,
(Origanum 0.4 g kg-1 days A. (Cyprinus carpio) ↑ Weight gain ↑ Skin mucus total Ig and performance Ghafarifarsani (65)
majorana) hydrophila ↓ FCR ↑ Skin mucus lysozyme activity
challenge ↑ SGR ↑ Skin mucus alternative complement
↑ Survival (ACH50) activity
against A.
hydrophila
Thyme Essential oils 500 ppm 30 days + Thymol, p-cymene and g- African catfish N/I ↓ Gill histopathologic lesions Mitigate the thiamethoxam induced El Euony, Elblehi
(Thymus thiamethoxam terpinene (Clarias garipenus) toxicity (66)
vulgaris) exposure
Thyme Aqueous extract 5.0, 10.0 and 2 weeks + N/I Rainbow trout No effect ↑ Intestine antioxidant enzymes (SOD, Mitigate adverse effects of Hoseini and
(Thymus 20.0 g kg-1 oxytetracycline (Oncorhynchus CAT, GPx and GST) activity oxytetracycline and improve the fish Yousefi (67)
vulgaris) mykiss) ↓ Intestine levels of the oxidative immune responses
stress marker malondialdehyde
(Continued)Frontiers in Immunology | www.frontiersin.org
Firmino et al.
TABLE 1 | Continued
Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference
plant origin form dosage(s) administration components (≤ 3)
tested
Thyme Essential oils 0.1%, 0.5%, 15 days Thymol, o-cymene and carvacrol Nile tilapia N/I No effect upon the population of Stimulated the cellular components of Valladão, Gallani
(Thymus and 1% (Oreochromis beneficial Bacillus bacteria in the gut the non-specific immune response (68)
vulgaris) niloticus) without deleterious effects on the
general health of the fish or the
intestinal tract
Thyme Essential oils 0.005, 0.010 5 weeks Thymol, p‐cymene and Rainbow trout No effect No effect upon the allochthonous No toxic effects do not significantly Navarrete, Toledo
(Thymus and 0.02 g linalool (Oncorhynchus microbiota profile alter the intestinal contents bacterial (69)
vulgaris) kg-1 mykiss) populations
Spanish Essential oils 0.001, 0.002, 12 weeks Thymol, p‐cymene and carvacrol Gilthead seabream No effect ↑ Anterior intestine lymphocyte Dose-dependent immuno‐modulatory Hernandez, Garcia
thyme 0.003 and (Sparus aurata) aggregates in the lamina propria at effect upon the intestine (70)
(Thymus 0.004 g kg-1 low dose
zygis subsp. ↓ Anterior intestine lymphocyte
gracilis) aggregates in the lamina propria at
high doses
Lemon balm Ethanolic extract 0.2% and 60 days + 7 N/I Nile tilapia ↑ Final weight ↑ Skin mucus total Ig Can effectively improve the fish Mohammadi,
(Melissa 0.5% days A. (Oreochromis ↑ Weight gain ↑ Skin mucus lysozyme activity growth, health, and immune status Rafiee (58)
officinalis) hydrophila niloticus) ↓ FCR ↑ Skin mucus protease activity
challenge ↑ SGR ↑ Skin mucus alternative complement
↑ Survival rate (ACH50) activity
↑ Survival
against A.
hydrophila
Peppermint Powder 2.0, 3.0, and 8 weeks N/I Caspian roach ↑ Final weight ↑ Secretion of skin mucosal protein Act as a growth promoter and Paknejad, Hosseini
(Mentha 4.0 g kg-1 (Rutilus caspicus) ↑ Weight gain pattern bands; higher lysozyme band immunostimulant Shekarabi (71)
piperita) ↓ FCR intensity in particular
↑ SGR ↑ Skin mucus lysozyme activity
↓ Daily intake ↑ Skin mucus alkaline phosphatase
rate activity
↑ Skin mucus soluble protein
Peppermint Essential oils 0.1 and 0.25 g 7, 14, 30 and Menthol, mentone and 1,8- Nile tilapia N/I ↑ Intestine intraepithelial lymphocytes Show benefits in terms of intestinal Valladão, Gallani
6
(Mentha kg-1 60 days cineole (Oreochromis health and on immune parameters (72)
piperita) niloticus)
Peppermint Ethanolic extract 1.0%, 2.0% 8 weeks N/I Rainbow trout ↑ Survival ↑ Skin mucus antibacterial activity Triggers the immune system of Adel, Pourgholam
(Mentha and 3.0% (Onchorhynchus against A. against S. iniae, Y. ruckeri, A. rainbow trout against Y. ruckeri (73)
piperita) mykiss) hydrophila hydrophila and L. garviea
Peppermint Ethanolic extract 1.0%, 2.0% 56 days N/I Caspian kutum ↑ Weight gain ↑ Skin mucus antibacterial activity Increases the mucosal immune Adel, Amiri (74)
(Mentha and 3.0% roach (Rutilus frisii ↓ FCR against S. iniae, Y. ruckeri, L. parameters and performance of fry in
piperita) kutum) ↑ SGR monocytogenes and E. coli a dose dependent manner
↑ Skin mucus protein level
↑ Skin mucus alkaline phosphatase
activity
Peppermint Ethanolic extract 1.0%, 2.0% 8 weeks N/I Caspian brown trout ↑ Weight gain ↑ Skin mucus protein level Promote growth performance and Adel, Safari (75)
(Mentha and 3.0% (Salmo trutta ↓ FCR ↑ Skin mucus lysozyme activity have immunostimulant properties
piperita) caspius) ↑ SGR ↑ Skin mucus alkaline phosphatase
activity
Horsemint Ethanolic extract 2.0%, 4.0% 8 weeks + Y. N/I Caspian kutum ↑ Weight gain ↑ Skin mucus protein level Improve growth performance and Gholamhosseini,
(Mentha and 6.0% ruckeri challenge roach (Rutilus frisii ↓ FCR ↑ Skin mucus lysozyme activity boost fish immune response in a Adel (76)
longifolia) kutum) ↑ SGR ↑ Skin mucus alkaline phosphatase dose‐related manner
June 2021 | Volume 12 | Article 695973
↑ Survival rate activity
Phytogenics and Fish Mucosal Immunity
↑ Skin mucus protease activity
↑ Skin mucus esterase activity
↑ Skin mucus antibacterial activity
against S. iniae, Y. ruckeri, A.
hydrophila and L. garvieae
Horsemint Hydroalcoholic 0.1%, 0.2% 4 weeks + 10 N/I Rainbow trout ↑ Survival ↑ Secretion of skin mucosal protein Dose‐related positive effect on Heydari,
(Mentha extract and 0.3% days Y. ruckeri (Onchorhynchus against Y. pattern bands; higher lysozyme band immunogenicity and increased Firouzbakhsh (77)
longifolia) challenge mykiss) ruckeri intensity in particular resistance to bacterial disease
Thumbai Powder 1.0, 2.0, 4.0 45 days + 15 N/I Nile tilapia ↑ Weight gain ↑ Skin mucus lysozyme activity Increase skin mucosal immune Kurian, Van Doan
(Leucas and days S. (Oreochromis ↓ FCR ↑ Skin mucus peroxidase activity parameters, performance and survival (78)
aspera) 8.0 g kg-1 agalactiae niloticus) ↑ SGR against bacterial infection
challenge ↑ Survival
(Continued)Frontiers in Immunology | www.frontiersin.org
Firmino et al.
TABLE 1 | Continued
Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference
plant origin form dosage(s) administration components (≤ 3)
tested
against S.
agalactiae
Shirazi Hydroalcoholic 2.0 g kg -1
56 days Thymol and carvacrol? (N/I) Rainbow trout ↑ Survival rate ↑ Skin mucus bactericidal activity Increase skin mucosal immunity Mirghaed, Hoseini
thyme extract (Oncorhynchus against A. hydrophila (79)
(Zataria mykiss) ↑ Skin mucus lysozyme activity
multiflora)
Shirazi Powder (1:1) 40 g kg-1 12 weeks N/I Common carp No effect No effect Do not prevent intestinal tissue lesions Tasa, Imani (80)
thyme + aflatoxin B1 (20 g kg-1 (Cyprinus carpio) induced by aflatoxin B1
(Zataria each)
multiflora)
+ Rosemary
(Rosmarinus
officinalis)
Rosemary Aqueous extract 10, 20, 40, 80 20 days 1,8-Cineole Common carp N/I ↑ Skin mucus level of 1,8-Cineole High volume of extracts might Zoral, Ishikawa
(Rosmarinus and 100 ml/ (Cyprinus carpio) dose-dependent promote hepatic toxicity (81)
officinalis) 100 g No effect upon intestine
histopathology
Rosemary Powder 0.6, 1.2, 1.8 4 and 12 weeks Carnosic acid and carnosol (1:1) Gilthead seabream No effect No effect The histological examination of the Hernandez, Garcia
(Rosmarinus and 2.4 g kg-1 (Sparus aurata) intestine showed no aspects that Garcia (82)
officinalis) might pose problems for absorption,
or any immune system disorder
associated with the intestine
Oliveria Essential oils and/ 0.01%, 0.1% 60 days + 14 g-terpinene, Nile tilapia ↑↓ Survival No effect Increase fish survival 14 days after Vazirzadeh, Jalali
(Oliveria or hydroethanolic and 1.0% days S. iniae carvacrol and thymol (Oreochromis against S. iniae challenge with S. iniae (83)
decumbens) extract challenge niloticus)
Clove basil Ethanolic extract 5.0, 10.0, and 12 weeks + 14 N/I African catfish ↑ final weight ↑ intestine villus length Improve the fish performance, health, Abdel-Tawwab,
(Ocimum 15.0 g kg-1 days L. (Clarias gariepinus) ↑ weight gain ↑ intestine villus width and immune response Adeshina (84)
gratissimum) monocytogenes ↑ SGR ↑ intestine absorption area
challenge ↑ Feed intake
↑ survival
7
against L.
monocytogenes
Clove basil Essential oils 0.5%, 1.0% 55 days + 10 1,8-cineole, eugenol and b- Nile tilapia N/I ↑ Intestine villus height Ameliorate tissue damages, even in Brum, Cardoso
(Ocimum and 1.5% days S. selinene (Oreochromis ↑ Intestine goblet cells number situations of infection (85)
gratissimum) agalactiae niloticus) ↓ Gill epithelial detachment in the
challenge secondary lamellae
↓ Gill congestion at the base of the
secondary lamellae
American Essential oils 0.25, 0.5, 1.0 7 weeks Linalool, eugenol and 1,8-cineole Red drum No effect ↑ Stomach lysozyme activity Different supplementation levels do not Sutili, Velasquez
basil and 2.0 g kg-1 (Sciaenops No effect upon the intestinal microbial influence growth performance and (86)
(Ocimum ocellatus) community intestinal microbial community;
americanum) however, show effects on
immunological responses
Savory Powder 1% 45 days N/I Common carp N/I ↑ Intestinal lactic acid bacteria Improves intestinal health Mousavi,
(Satureja (Cyprinus carpio) Mohammadiazarm
khuzestanica) (87)
Allium sp.
Garlic Aqueous extract 0.10, 0.15, and 80 days N/I Guppy (Poecilia No effect ↑ Skin mucus lysozyme Administration of 0.15 mL of garlic Motlag, Safari (88)
June 2021 | Volume 12 | Article 695973
(Allium 0.20 ml kg-1 reticulata) ↑ Skin mucus alternative complement extract per kg feed is suggested to
Phytogenics and Fish Mucosal Immunity
sativum) ↑ Skin mucus total Ig obtain optimal skin mucus immunity
↑ Skin mucus alkaline phosphatase
Garlic Oil 50 µl kg-1 28 days + N/I Rohu (Labeo rohita) N/I ↓ Gill oxidative stress enzymes activity Amelioration of silver nanoparticles- Khan, Qureshi (89)
(Allium exposure to ↓ Gill histopathologic lesions induced oxidative stress and
sativum) silver histoprotective effects
nanoparticles
Garlic Powder 0.5 g and 1.0 2 months + N/I Nile tilapia ↑ Survival ↑ Anterior intestine transcriptional Could be effective in the prevention of Foysal, Alam (90)
(Allium g/100 g 2 weeks S. iniae (Oreochromis against S. iniae levels of interleukin genes (IL‐10 and S. iniae infection in fish
sativum) challenge niloticus) IL‐17F)
↑ OTU counts for the phylum of
Proteobacteria and Tenericutes
Garlic Powder 5.0%, 10.0% 14 or 28 or 32 Allicin (1.25 mg/g) Guppy (Poecilia ↓↑ Gills and N/I No clear preventative effect against C. Kim, Fridman (91)
(Allium and 20.0% days + C. reticulata) caudal fin C. irritans
sativum) irritans challenge irritans infection
(Continued)Frontiers in Immunology | www.frontiersin.org
Firmino et al.
TABLE 1 | Continued
Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference
plant origin form dosage(s) administration components (≤ 3)
tested
Garlic Powder 1.0%, 1.5% 120 days Rainbow trout ↑ Weight gain ↓ Bacterial diversity and richness Beneficial in terms of promoting Büyükdeveci,
(Allium and 2.0% (Oncorhynchus ↑ SGR ↑Deefgea, Mycoplasma, growth and inducing changes in the Balcá zar (92)
sativum) mykiss) Exiguobacterium and Clostridium intestinal microbiota in a dose-
genera dependent manner
↓↑ Aeromonas genus
Garlic Powder 5.0, 10.0 and 8 weeks N/I Caspian roach ↑ Weight ↑ Skin mucus antibacterial activity Beneficially affects the skin mucus Ghehdarijani,
(Allium 15.0 g kg-1 (Rutilus rutilus) ↑ Growth rate against S. faecium, M. luteus, S. immune parameters and growth Hajimoradloo (93)
sativum) diet marcescens and E. coli performance
↑ Skin mucus protein level
↑ Skin mucus alkaline phosphatase
activity
Garlic Lyophilized 2.0% 21 days + N/I Prussian carp N/I ↓ Gill histopathologic lesions Shows chelating and antioxidant Nicula, Dumitrescu
(Allium cadmium (Carassius gibelio) potential (94)
sativum) exposure
Onion (Allium Powder 1% 45 days N/I Common carp N/I ↑ Intestinal lactic acid bacteria Improves intestinal health Mousavi,
cepa) (Cyprinus carpio) Mohammadiazarm
(87)
Onion (Allium Ethanolic extract 0.5%, 1.0%, 12 weeks N/I African sharptooth No effect ↓ Intestine villus length Increase the digestive and absorptive Bello, Emikpe (95)
cepa) 1.5% and catfish (Clarias ↑ Intestine villus width capacity of the intestine
2.0% gariepinus) ↑ Intestine absorption area
↑ Intestine cryptal depth
Mongolian Ethanolic extract 0.04 g kg-1 4 weeks + Flavonoids ≥90% (HPLC) Grass carp N/I ↓ Intestine and gill Cr accumulation Decrease in Cr-accumulation, Zhao, Yuan (96)
Wild Onion chromium (Cr) (Ctenopharyngodon ↓ Gill malondialdehyde content oxidative stress, immunosuppression
(Allium exposure idella) ↓ Gill protein carbonyl content and inflammatory response following
mongolicum) ↑ Intestine lysozyme activity Cr exposure
↑ Intestine complement 3 levels
↑ Intestine and gill tight junction
proteins gene expression
↑↓ Intestine and gill NF-kB signaling
pathway gene expression
8
Single bioactive compounds
Thyme Ethanolic solution 0.15, 0.3, 0.45, 56 days + 14 Thymol (commercial product) Snakehead fish ↑ Final weight ↑ Intestine SOD, CAT, GSH-Px Adequate dietary supplementation can Kong, Li (97)
(Thymus 0.6, 0.75 g kg- days A. veronii (Channa argus) ↑ Weight gain activities effectively enhance the growth,
vulgaris)* 1
challenge ↑ SGR ↓ Intestine malondialdehyde content antioxidant status, immune response
↑ Protein ↑ IL-10 and TGF-b gene expression in and disease resistance
efficiency ratio intestine
↓ FCR ↓ HSP70, TNF-a, IL-1b and IL-8 gene
↑ Survival to A. expression in intestine
veronii
Thyme N/I 0.1, 0.2 and 60 days + 8 Thymol (commercial product) Grass carp ↑ Final weight ↑ Gill enzymes of the phosphotransfer Favors weight gain and fish longevity. Morselli, Baldissera
(Thymus 0.3 g kg-1 days A. (Ctenopharyngodon ↑ Survival network: cytosolic and mitochondrial Prevents A. hydrophila induced (98)
vulgaris)* hydrophila idella) against A. creatine kinases, adenylate kinase branchial bioenergetics. High
challenge hydrophila activities and ATP levels in infected concentrations deserve attention
fish because of side-effects
↓ Gill ROS levels in infected fish
Thyme Crystals 0.5 g kg-1 70 days Thymol (99% purity; commercial Nile tilapia ↑ SGR ↓ Intestine total aerobic and anaerobic Improve some performance El-Naby, Al-
(Thymus product) (Oreochromis ↑ Protein counts parameters and negatively modulates Sagheer (99)
vulgaris)* niloticus) efficiency ratio intestinal microbial communities.
June 2021 | Volume 12 | Article 695973
Demonstrates a notable synergistic
Phytogenics and Fish Mucosal Immunity
interaction with chitosan nanoparticle
with beneficial effects
Thyme N/I 1.0 g kg-1 56 days Thymol (commercial product) Rainbow trout ↓ FCR ↓ Intestine culturable anaerobe Modulated intestinal microbial Giannenas,
(Thymus (Oncorhynchus bacteria communities disfavoring total Triantafillou (100)
vulgaris)* mykiss) anaerobes
Oregano N/I 1.0 g kg-1 56 days Carvacrol (commercial product) Rainbow trout ↓ FCR ↓ Intestine culturable anaerobe Modulated intestinal microbial Giannenas,
(Origanum (Oncorhynchus bacteria communities disfavoring total Triantafillou (100)
vulgare)* mykiss) anaerobes
Chinese Powder 0.4, 0.8 and 60 days + H2O2 Baicalin (80% purity, commercial Nile tilapia ↓ FCR ↑ Gill glutathione level Improves feed efficiency, enhance Jia, Du (101)
skullcap 1.6 g kg-1 challenge product) (Oreochromis ↑ Gill total antioxidant capacity antioxidative ability and alleviate
(Scutellaria niloticus) oxidative stress
baicalensis)*
Garlic Liquid 0.005%, 30 days Allicin (98% purity, commercial Large yellow ↑ Final weight ↑ Intestine total antioxidant capacity Improve the survival and growth of Huang, Yao (102)
(Allium 0.01% and product) croaker ↑ Final length ↑ Intestine antioxidant enzymes (CAT, large yellow croaker larvae probably
sativum)* 0.02% NO and NOS) activity by promoting intestinal development,
(Continued)Frontiers in Immunology | www.frontiersin.org
Firmino et al.
TABLE 1 | Continued
Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference
plant origin form dosage(s) administration components (≤ 3)
tested
(Larimichthys ↑ SGR ↓ Intestine transcriptional levels of pro- alleviating inflammation and enhancing
crocea) ↑ survival rate inflammatory genes appetite
Phytogenics combinations
Phytogenics Essential oils 200 ppm 70 days + 15 Garlic and Lamiaceae-plants oils Greater amberjack No effect ↑ Skin mucus lysozyme activity 15 Facilitates the immunological response Ferná ndez-
combination days N. girellae (N/I; commercial additive) (Seriola dumerili) days post N. girellae challenge of skin once the parasite is fixed, Montero,
challenge ↑ Piscidin gene expression in skin pre- generating a hostile microenvironment Torrecillas (16)
challenge in skin and lowering the parasite load
↑ Proinflammatory cytokines (tnf-a
and il1-b), AMPs (hep and cath),
immunoglobulin (IgT), complement
protein (c3) T-cells marker (cd8) and
mucin (muc-2) gene expression in skin
post-challenge
↓ Casp3 gene expression in skin post-
challenge
Phytogenics Essential oils 0.3 g kg-1 8 weeks + 2 Cinnamaldehyde, thymol and Nile tilapia ↓ ↑ Intestine villi density Positive effects of digestion and Ning, Zhang (103)
combination weeks hypoxia carvacrol (Oreochromis Hepatosomatic ↓ Intestine malondialdehyde content antioxidative capacity
challenge niloticus) index
(Total 10 weeks)
Phytogenics Microencapsulated 0.5% 65 days Garlic essential oil (N/I), carvacrol Gilthead seabream No effect ↑ Skin mucus inhibitory activity against Beneficially affects the skin and mucus Firmino,
combination essential oils and thymol (Sparus aurata) V. anguillarum and P. anguilliseptica immune and stress parameters, Ferná ndez-Alacid
(Commercial additive) ↓ Cortisol in skin mucus suggesting the stimulation and (14)
↑ Regulation of genes associated to recruitment of phagocytic cells and a
the secretory pathway in skin reduction in the fish allostatic load
↑ Regulation of genes associated to
non-specific immune response in skin
↑ Regulation of genes coding for
oxidative stress enzymes in skin
Phytogenics Microencapsulated 0.5% 65 days Garlic essential oil (N/I), carvacrol Gilthead seabream No effect ↑ Regulation of genes related to The activation of leukocytes and Firmino, Vallejos-
combination essential oils and thymol (Sparus aurata) processes of proteolysis and crosstalk between gut and microbiota Vidal (12)
9
(Commercial additive) inflammatory modulation, immunity, are suggested to regulate the
transport and secretion, response to inflammatory response induced by the
cyclic compounds, symbiosis, and additive
RNA metabolism in the mid-anterior
intestine
No effect upon alpha diversity of
bacterial communities in the anterior
and posterior intestinal tract sections
↓ Spirochaetes phylum in the posterior
intestine
↑ Photobacterium and
Corynebacterium genera in the
anterior intestine
↓ Comamonas in the anterior intestine,
and Paracoccus, Prevotella and
Rothia genera in the posterior intestine
↓ Bacterial sequences related to
carbohydrate and drug metabolisms,
June 2021 | Volume 12 | Article 695973
and membrane transport
Phytogenics and Fish Mucosal Immunity
↑ Bacterial sequences related to
glutathione and lipid metabolisms,
naphthalene degradation and sulphur
relay system
Evidence of host-microbial co-
metabolism
Phytogenics Microencapsulated 0.5% 65 days + 39 Garlic essential oil (N/I), carvacrol Gilthead seabream ↓ S. ↑ Regulation of genes related pro- Promotes gill mucosal immunity and Firmino, Vallejos-
combination essential oils days S. and thymol (Sparus aurata) chrysophrii total inflammatory immune response reduces gill ectoparasite incidence Vidal (15)
chrysophrii (Commercial additive) parasitation arbitrated by degranulating acidophilic
granulocytes, sustained by antioxidant
(Continued)Frontiers in Immunology | www.frontiersin.org
Firmino et al.
TABLE 1 | Continued
Phytogenic Supplemented Inclusion Period of Main bioactive Fish species Performance Mucosal parameters evaluation Key benefits summary Reference
plant origin form dosage(s) administration components (≤ 3)
tested
challenge (total and anti-inflammatory responses in
104 days) gills
↑ Carboxylate glycoproteins containing
sialic acid in mucous and
epithelial gill’s cells
Phytogenics Essential oils 0.02% 9 weeks + 1 Garlic and Lamiaceae-plants oils European sea bass ↑ Survival ↑ Skin mucus lysozyme activity when Attenuate the fish physiological Serradell,
combination week stress and (N/I; commercial additive) (Dicentrarchus against Vibrio stress and bacterial challenge response to stress increasing Torrecillas (104)
V. anguillarum labrax) anguillarum resistance to Vibrio anguillarum
challenge when stress- infection
challenged
Phytogenics Essential oils 0.02% 63 days Garlic and Lamiaceae-plants oils European sea bass No effect ↓ Shannon alpha diversity of mucosa- Reduction of orders containing Rimoldi, Torrecillas
combination (N/I; commercial additive) (Dicentrarchus associated microbiota potentially pathogenic species for fish, (105)
labrax) ↑Clostridiales order in intestinal and enrichment of gut microbiota
content composition with butyrate producer
↓ Coliforms and Vibrionales taxa
allochthonous microbiota
Phytogenics Essential oils 200 ppm 63 days + 7 Garlic and Lamiaceae-plants oils European sea bass No effect ↓ Posterior intestine fold area covered Protective effect focused mainly on the Torrecillas, Terova
combination days stress and (N/I; commercial additive) (Dicentrarchus by goblet cells preileorectal valve region (13)
V. anguillarum labrax) ↓ Posterior intestine goblet cells area
challenge ↑ Intestine mucus coverage post-
challenge
Phytogenics N/I extract 6.0 g kg-1 30 days + 10 Saint John’s wort (Hypericum Atlantic salmon No effect ↓ Gut lipid peroxidation Improves the gut antioxidant status Reyes-Cerpa,
combination days crowding perforatum, Hypericacea), lemon (Salmo salar) Vallejos-Vidal (106)
stress (40 days balm (Melissa officinalis,
total) Lamiaceae) and rosemary
(Rosmarinus officinalis,
Lamiaceae) mixed at a ratio 3:2:1
Phytogenics Powder 1% 45 days Savory (Satureja khuzestanica, Common carp N/I ↑ Intestinal lactic acid bacteria Improves intestinal health Mousavi,
combination Lamiaceae) 0.5% and Onion (Cyprinus carpio) Mohammadiazarm
(Allium cepa, Alliaceae) 0.5% (87)
10
Phytogenics Essential oils 0.06, 0.2, 0.4 6 weeks Thymol and carvacrol (1:1; Hybrid tilapia (O. N/I ↑ Posterior intestine villus height Affect the immunity primarily through a Ran, Hu (107)
combination and 0.8 g kg-1 commercial additive) niloticus ♀ × O. ↑ Posterior intestine goblet cell count direct effect on host tissue but also
aureus ♂) per villus has an indirect effect mediated by
↑ Anterior intestine intraepithelial microbial changes
leucocytes
↓ Distal intestine intraepithelial
leucocytes
↑ OTUs, and PD whole tree and
Chao1 diversity indexes
↓Thermi phylum and Bacteroides,
Candidatus Cardinium, and
Leptospirillum genera
Phytogenics Essential oils 100 ppm 9 weeks 25% thymol and 25% carvacrol Gilthead seabream ↓ FGR ↑ Intestine mucosal foldings Induce an anti-inflammatory and anti- Perez-Sanchez,
combination (commercial additive) (Sparus aurata) ↑ Intestine enterocytes proliferative transcriptomic profile with Benedito-Palos
↑ Intestine goblet cells probable improvement in the (108)
↓ Expression of genes related to cell absorptive capacity of the intestine
differentiation and proliferation,
intestinal architecture and
June 2021 | Volume 12 | Article 695973
permeability, immunosurveillance,
Phytogenics and Fish Mucosal Immunity
such as cytokines, in the intestine
SGR, Specific Growth Rate.
FCR, Feed Conversion Ratio.
FGR, Feed Gain Ratio.
OTU, Operational Taxonomic Unit.
*Indicate the putative plant species with high content of the referred bioactive compound. N/I, not identified or not assessed.
Studies reporting the application of plant extracts or related compounds as bath treatments or evaluating bactericidal or antiparasitic effects in vitro were excluded from the selection. The table omits systemic immunity-related results, digestive
enzymes or other complementary analysis performed within each study. Blends with other components besides the selected group of plants, terpenes or organosulfur compounds were excluded as well.Firmino et al. Phytogenics and Fish Mucosal Immunity
mucus, few studies have tried to explain and characterize the conditions and acquire relevant data for their further
immunomodulatory mechanisms underlying such responses development and general and safe use.
neither which specific compounds might be exerting such effects. The effect of some single specific bioactive compounds related
Carvacrol, thymol, p-cymene and g‐terpinene are identified as to aromatic plants of the Lamiaceae family, such thymol or
the predominant bioactive compounds of most of the members carvacrol, upon mucosal tissues were also reported (Table 1).
of the Lamiaceae family considered in this review, which were Although these studies have the advantage to associate a specific
mainly found in oregano and thyme. In addition, peppermint, mucosal response to the administration of a specific compound,
rosemary and basil contain preponderant concentration of other once again it is observed that most of them only reported the
bioactive compounds such menthol, eugenol and 1,8-cineole analysis of few immune and oxidative markers (97, 98, 101, 103),
(Table 1). Interestingly, some studies have reported carvacrol or a limited microbiological examination (99, 100). In fact, few
and 1,8-cineole presence in the fish skin mucus (63, 81). This studies were observed to apply complementary analysis, or
phenomenon of bioactive compounds efflux through skin could achieved to successfully discuss the multifactorial impact
be responsible for the immunomodulatory and antimicrobial exerted by such nutritional strategies upon mucosal tissues
effects observed in the fish skin mucus. However, most of the (107, 108).
reviewed studies did not report the phytogenics composition Although the overall data suggest the therapeutic potential of
neither the assessment of their translocation through mucus. phytogenics derived from Lamiaceae family of plants in
Some studies have also reported a protective effect through aquafeeds, especially of their associated terpene phenolic
the reduction in gills’ histopathological lesions induced by toxic compounds, unfortunately none of the studies has proposed
element exposure or pathogenic challenges (62, 66, 85). accurate mechanisms that could be responsible for the broad
Contrarily to the studies describing the effects of phytogenics effects of these metabolites described upon fish mucosal tissues.
upon skin mucus secretion and their immunomodulatory Despite the lack of reliable information for aquaculture relevant
potential, their impact upon the GIALT is very scarce, being fish species, it is possible that the above-mentioned antimicrobial
mostly limited to histological observations. Similarly, studies on properties of these compounds, their free radicals’ scavenging
the impact of phytogenics upon the intestine are commonly ability, along with their aptitude to activate TRP channels that
focused on evaluating alterations in morphoanatomical modulate inflammatory processes may underlie the
parameters such as an increase in villus length, width and immunomodulatory properties and microbiota modulation
goblet cells count, which are usually associated to described in different mucosal tissues.
improvements in fish growth performance (59, 60, 64, 84, 85).
Some studies have also reported the modulation in the number of Effect of Dietary Organosulfur Compounds
intestinal lymphocytes (70, 72). Other authors have described a Upon Fish Mucosal Immunity
positive impact upon the gastrointestinal activity of humoral The main constituents of extracts and essential oils from Allium sp.,
immune markers, such lysozyme (86), the activity of antioxidant such as garlic (Allium sativum, Alliaceae) and onion (Allium cepa,
enzymes and oxidative markers (67, 106) or the down-regulation Alliaceae), are sulfur‐containing compounds. This group of
of the expression of pro-inflammatory genes, such tnfa and tnfb bioactive substances comprises alliin, allicin and its derived
(61). Besides, the beneficial impact of phytogenics administration bioactive compounds like ajoene, diallyl trisulfide (DATS), diallyl
upon the intestine microbiota composition was also suggested disulfide (DADS), diallyl sulfide (DAS) and allyl methyl disulfide,
(61, 87). Nonetheless, analysis described were constantly commonly termed as organosulfur compounds. These organosulfur
incomplete in terms of mucosal immune response evaluation, compounds are the responsible for the recognized antioxidative,
since limited classical immune or oxidative markers were antimicrobial, antifungal and antiparasitic properties of garlic (126).
assessed in each of the above-mentioned studies. Similarly, Allicin (S-allyl-2-propenyl thiosulfinate) is usually the main
microbiota studies were often restricted to a particular group biologically active component of garlic and related species;
of bacteria, such lactic bacteria, failing to properly characterize however, it is highly unstable under physiological conditions;
microbiota functionality and modulation by the experimental thus, quickly being transformed into its organosulfur derivates,
diets; thus, resulting in partial and biased conclusions when which also exhibit therapeutic properties (127–129).
assessing the regulatory effects of functional feed additives on Organosulfur compounds have been particularly studied for
mucosal tissues. their antiparasitic activity. Among them, ajoene was described to
In addition, some studies reported no effect of the administrated interfere with parasite and host cell membrane protein and lipid
phytogenics upon mucosal parameters (68, 69, 80, 82, 83). Such trafficking, with irreversible detrimental consequences for the
discrepancies among studies evaluating a particular plant extracts parasite (130). This is of special relevance since teleost mucosal
may be due to the diversity of the referred studies in terms of tissues are known to have a high constitutive expression of Th2
experimental design, fish species selected, plant origin, markers that indicate a skewed immune response targeted
supplemented form and inclusion level of phytogenetics, among against parasites (131). Regarding their bactericidal properties,
others. This miscellaneous of studies and the lack of protocols the organosulfur compounds can penetrate the bacterial cell
allowing appropriate additive and animal testing, highlights the membranes, cause changes in the structure of thiol (-SH)
urgent need to standardize the experimental designs and procedures containing enzymes and proteins, and lower the expression of
in order to properly evaluate these compounds under in vivo important genes involved in the QS in bacteria, inhibiting the
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