Sustainable food supply to feed the demand of the projected world population by 2050 is a challenge, in which aquaculture is predicted to be the main source of aquatic dietary proteins. Such growth will unavoidably involve the implementation of innovative aquaculture production strategies, targeting issues related to effective health management and animal welfare (1). In this regard, the development and application of functional feeds represent a sound strategy to improve aquaculture systems, since they provide functional health benefits to animals beyond their nutritional value (2). In this scenario, phytogenics, also known as phytobiotics, are defined as environmentally friendly plant-derived bioactive compounds used as functional feed additives that show positive effects on animal growth and health. Phytogenic often comprise aromatic plants extracts, and essential oils characterized by its richness in biologically active compounds (3, 4). In farmed fish, a wide spectrum of phytogenics have been increasingly studied mainly due to their wide repertoire of properties, including growth promotion, and antimicrobial, immunostimulant, antioxidant, anti-inflammatory and sedative activities (5). In particular, phytogenics derived from Lamiaceae family and Allium sp. are among the most commonly studied and administrated plant-based additives (6, 7). Nonetheless, the complexity of the mechanisms of action and the pharmacological effects promoted by the diverse bioactive compounds present in such plants, along with their frequently observed synergistic behavior (8), often limits the full understanding of their biological activity (9).

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 global scale. The impact of phytogenics upon fish systemic immunity has been extensively tested in the past (5, 9). However, an increasing trend to evaluate phytogenics’ impact upon the mucosal immunity has been gained importance in recent years, which is mainly attributed to the importance of the mucosal barrier in the fish defense against variated threats and, potential colonization and invasion by pathogenic organisms (11). In contrast, most of the studies evaluating the effect of phytogenics in fish systemic immunity are only supported by a selection of repetitive primary analyses (i.e., lysozyme, bacteriolytic and complement activities, immunoglobulins, oxidative stress enzymes, etc.) serving only as proxies, that only provide a snapshot of the effects of the evaluated feed additive on the organism. These approaches do not allow elucidating their mode of action at cellular and molecular levels. This is of special relevance when dealing with functional feed additives with potential pharmacological properties, as their standardized use mainly depends on the proper understanding of their regulatory properties in the immune system.

Phytogenic administration has the potential to regulate the mucosal barrier function by means of several molecular mechanisms, in which the phytogenic bioactive compounds interact with cellular transcription factors and metabolic cascades. Therefore, the modulation of the expression of genes coding for immune relevant molecules alter the mucosal protective characteristics and their immunological status (12–16). Besides, the immune system influences the regulation and composition of the microbiota and vice versa, an interaction that plays a determinant role in the maintenance of the mucosal integrity and functionality (17, 18). Hence, both the improvement of the mucosal barrier characteristics and the modulation of the microbiota are central targets for the development of new phytogenic additives, while understanding their mode of action at cellular and molecular levels is critical for elucidating their benefits to the host.

Given the extended literature available on plant-based functional additives and the significance of the mucosal immunity described above, our review efforts focus exclusively on the physiological and immunological responses achieved by the most studied fish mucosal tissues, intestine, gills and skin, of organisms fed with phytogenics of the Lamiaceae family and Allium sp. In the first part, we present a thorough description on their main bioactive compounds and relevant biological properties. Then, the immunomodulatory properties and the mechanisms they can trigger on the fish mucosal tissues are explored and further potential mechanisms hypothesized. Finally, research gaps and constrains for the development of applicable phytogenic-based additives are discussed. Overall, the information presented herein aims to provide clear insights on the fish mucosal immune response dietary treated with phytogenics, propose viable mechanisms for exploiting them, and ultimately encourage the practice of reliable and reproducible research for the development of novel feed additives to be used as sustainable and safe prophylactic strategies for aquaculture.

The mucosal barrier constitutes the fish first line of defense against the surrounding environment and potential pathogens. Fish mucosal tissues are particularly characterized by a mucosa-associated lymphoid tissue (MALT), harboring diverse myeloid and lymphoid cells that are responsible for the host protection against pathogens and antigens, while tolerating beneficial symbiont colonization to maintain mucosa homeostasis (19–21). Six different MALTs have been described so far in teleosts. The gut-associated lymphoid tissue (GALT), the gill-associated lymphoid tissue (GIALT), the skin-associated lymphoid tissue (SALT), the nasopharynx-associated lymphoid tissue (NALT) and, the more recently characterized the buccal, and pharyngeal MALTs (22). Other mucosal immune systems have been hypothesized and are currently under study (23). Despite the existence of others, the GALT, GIALT and SALT are the most studied and well characterized MALTs and therefore selected as target in this review.

Among the extensive cell types with immune capacity coexisting in the fish body, upon sensing the presence of pathogenic or commensal microbe-associated molecular pattern (MAMP) a downstream signaling response mediated through pattern recognition receptors (PRRs) immediately takes place. So far, several piscine PRRs have been identified, being the toll-like receptors (TLR), NOD-like receptors (NLR) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) the best characterized (24, 25). Epithelial and endothelial cells together with the professional phagocytes, represented by macrophages, granulocytes and dendritic cells, are the first responders against MAMPs formerly sensed by PRRs. Phagocytosis contributes to both pathogen clearance and subsequent antigen presentation to other immune cells by the membrane Major Histocompatibility Complex (MHC) class II peptide complex (26). In most, but not all teleost fish, the peptide-MHC II complex activates naive CD4⁺ T cells expressing antigen-specific T cell receptors (TCR) in their surface. Recognition of this complex stimulates the dedicated CD4⁺ cells activation and differentiation into T helper cell subsets possessing inflammatory cytokines secreting capacity that further coordinate the adaptive response together with B cells (27, 28). Interestingly, while CD4⁺ T helper lymphocytes are mainly present in the gut lamina propria, the cytotoxic CD8⁺ cells are the dominant intraepithelial resident immunocytes (29–31).

In fact, both T and B lymphocytes are abundantly present in fish mucosal tissues (32). Interestingly, the phagocytic and bacterial-killing abilities of B cells in fish have been fairly introduced in the past (33). However, their antigen presentation mechanisms mediated by MHC II and costimulatory molecules (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 mucosal immunoglobulin analogous to mammalian IgA (36), plays a critical role in the clearance of mucosal pathogens and the preservation of microbiota homeostasis through immune exclusion (11). Although extremely important in mucosal defense, not all teleosts present IgT/IgD, which suggests the existence of alternative mucosal immune systems (23).

For instance, the complete IgM and IgT sequences in their membrane and soluble forms have been reported and characterized for the first time in a perciform, the gilthead sea bream (Sparus aurata) (37). Interestingly, this study demonstrated that virus and bacteria trigger the mucosal immune response by promoting the activation of IgT in seabream. Although, diets with fish oil replacement by ones from plant origin inhibited the IgT up-regulation upon intestinal parasitic challenge, which was related to a worse disease outcome. These results evidenced that mucosal immunoglobulins can be significantly affected by dietary treatments, which highlights the necessity of testing this response case by case.

Although characterized by common cellular components, immune mediators and immune mechanisms, the different composition, organization and functions of MALTs may vary according to each tissue intrinsic and external environmental factors (38), changes that may be associated to the fish species considered. Besides, the microbiota also stands as a relevant component of the mucosal barrier, displaying an antagonistic behavior against invading “hostile” microorganisms and directly participating in the immune responses through the complex host-microbiota crosstalk at the mucosal interface (17, 18). Therefore, the selective manipulation of the microbiota by means of nutritional approaches has been previously proposed as a viable alternative to modulate mucosal responses (39).

The mucosal tissues are intrinsically characterized by mucus secreting cells, such as goblet cells (40). Beside playing important roles in intra- and interspecific ecological interactions (41) and being a key component that ensures host-microbiota homeostasis (42, 43), the secreted semipermeable mucus represents the first challenge that pathogens have to overcome in order to interact with the host. Its complex composition encompasses a matrix of glycoproteins, the mucins that confer the mucus its structure, and a wide variety of humoral immune factors, such as lysozymes, complement, lectins, proteolytic enzymes, antimicrobial peptides, immunoglobulins, among others (41). Moreover, the mucus is continuously secreted and replaced (44); this continuous secretion aims to prevent pathogen attachment and interaction with the host. Therefore, the presence of a mucus layer is fundamentally involved in the regulation of the mucosal immune system, not only as a protective physical and chemical barrier, but also acting as a vehicle for mucins and humoral immune factors from the inside out. Both goblet cells (13, 15) and mucus composition (45) are highly susceptible to be manipulated through dietary strategies, which opens a wide range of possibilities when to design and apply new functional feed additives.

In nature, plant secondary metabolites have functional roles independent from plant growth and development; thus, protecting plants from herbivore and pests, or acting as chemoattractants for pollinators (46). These bioactive compounds broadly found in aromatic plants are usually present as mixtures, mainly represented by phenolics and terpenes that are chemically characterized by their aromatic rings (3). Therefore, their benefits as dietary supplements are subject to the variability and complexity of the aromatic compounds mixture, apart from their synergistic effect, their origin, the dietary inclusion level and their pharmokinetics (47).

In particular, phytogenics derived from Lamiaceae family and Allium sp. are among the most widespread administrated plant-based additives in aquaculture (48) and livestock (6, 7). These compounds are used for their recognized growth promoting, antimicrobial, immunostimulant, antioxidant, anti-inflammatory and sedative properties. Although they can be found worldwide, some representatives of this group of aromatic plants (i.e., oregano, thyme, basil, menthe, rosemary, sage, marjoram, garlic and onion, among others) are particularly present and traditionally consumed in the Mediterranean area and appreciated in terms of human nutrition and therapy (49, 50). The health-promoting properties of these aromatic plants have been extensively reviewed in different aquaculture species (5, 48, 51–55). However, most of the studies dealing with these functional feed additives were only focused in physiological or biochemical responses, but few of them have elucidated the cellular and molecular mechanisms underlying their immunostimulatory capacity.

While the existent information about the inherent effect of these phytogenics upon immune cells is limited under in vitro conditions, numerous in vivo studies have demonstrated an improvement of the fish mucosal immune responses following their administration. A refined complementary search through Web of Science, PubMed and Google Scholar was performed in this review. Until March 2021, 62 publications reporting the nutritional effects of Lamiaceae family and Allium sp., or related bioactive compounds, upon fish mucosal responses were retrieved and their results summarized in Table 1 . Importantly, most of them were published in the last year; thus, evidencing the current increasing interest for research on phytogenics targeting mucosal tissues. From the overall bibliographic search results, few publications felt within the objective of the present review and described the cellular or molecular mechanisms underlying fish mucosal immune responses to phytogenics’ administration. 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 as this review is just focused on the mucosal immune mechanisms. Furthermore, Table 1 omits those results out of the mucosal immunity context, including systemic immunity-related results, non-immune digestive parameters or other complementary analysis performed within each study. Although such variables are extensively used as key performance indicators in such studies, their relevance in terms of supporting and/or establishing the mode of action of phytogenics is questionable and out of the scope for this review. Finally, blends with other components besides the selected group of plants –or associated bioactive compounds such as terpenes or organosulfurs– were excluded as well.

Feeding the projected world population by 2050 in a sustainable way is a great challenge, in which aquaculture is predicted to supply the majority of aquatic dietary protein. For that, the implementation of novel policies and production system approaches targeting effective health management and animal welfare are mandatory (1). Moreover, indiscriminate prophylactic use of antibiotics associated to intensive aquaculture practices can still be observed among some of the major aquaculture producing countries, as it has been recently reviewed (148, 149). However, in 2022, several countries, including the EU will prohibit all forms of routine antibiotic use in farming, including preventative group treatments which highlights the necessity for the development of more sustainable alternative treatments (150). Under this context, the market for sustainable products and feed additives is increasingly growing and the number of studies on the use of a wide variety phytogenics as sustainable tools to be implemented in aquaculture production has increased dramatically in the last decade (5). The global market of phytogenic feed additives including major tier I and II suppliers was estimated on 753M USD in 2020 and it is projected to reach 1,098M USD by 2025 (151).

Although limitations in testing and reproducing studies using dietary immunostimulants have been pointed out since long ago (152–155), the current knowledge on the pathways and mechanisms followed by these compounds at the cellular level is still neglected. A large selection of experimental designs, fish species, phytogenics tested and diet composition fails in the association of selected bioactive compounds to specific effects. Moreover, it is important to consider the difficulties to carry out comparison among the available studies because of the different assays, testing methods, different extraction procedures of plant essential oils or extracts, and the intrinsic variation in chemical phytoconstituents in plants due to different agroclimatic conditions, harvesting season and plant phenotype. This essential oils or extracts consist of a variable mixture of different bioactive molecules that area generally not characterized, and are administrated through variable periods of time, dietary doses and forms in different fish species that are generally randomly selected. In addition, a considerable number of these studies provide little or partial information regarding the effect of a given immunostimulant, since their definitive efficiency assessment rely on the evaluation of basic biochemical parameters that are to some extent obsolete if compared against the actual state-of-the-art. Based on the former idea, diverse omics tools available play a fundamental role for proper understanding and characterization of their mode of action in mucosal tissues at cellular level. Together, these factors question the reproducibility and safety of a large number of studies available and limits the use of several of those proposed substances in commercial functional feeds.

In this regard, we propose that the study of purified bioactive compounds may represent a viable solution to circumvent variability, and the biological mode of action of isolated compounds should be primarily assessed in vitro under variated settings, before moving forward to in vivo trials. However, it is important to consider that the biological activities of essential oils or extracts cannot be attributed to a single compound or to a unique specific mechanism, since their multi-component properties exert greater biological activity when compared to the major components alone, whose function is probably regulated by the synergy with limiting compounds (8). In this light, essential oils and extracts from different plants have been explored for their potential as resistance modifying agents (156). While their chemical complexity may represent a clear advantage in terms of reducing the risk of inducing bacterial adaptation and resistance to single compounds, or even promoting a wider antibacterial activity, the use of blends of phytogenics makes difficult to proper characterize their mode of action. Therefore, when developing such additives, the formulation of combined purified compounds through the correct and soundly in vitro functional characterization to obtain potential synergies is recommended. Moreover, long-term studies assessing whether the bioactive compounds, single or combined, induce bacterial tolerance, transmissible adapted resistance or any other change on a large scale should be implemented and the effects on both beneficial and pathogenic bacteria determined through in vitro, in silico and finally in vivo approaches (103, 157).

While several phytogenics have been proved remarkably efficient in promoting mucosal fitness (9), little efforts have been made to elucidate the underlying pharmacokinetics and immunostimulatory mechanisms of tested compounds upon the MALTs, with few in vitro studies published to date. It should also be highlighted that occasionally in vitro studies do not accurately translate into predictable responses in vivo (83); thus, both in vitro and in vivo studies should be performed whenever possible. This lack of complementary information supports the demand for additional profound research on the fate and length in which particular phytogenic compounds act, which is crucial for further developing functional additives and their application in an industrial context. Although the specific mechanisms behind the observed fish mucosal physiological responses are still poorly described, it is possible that cellular pathways involving the activation of TRP receptors by the bioactive compounds might be responsible for the reported mucosal immune responses. Besides, this response might be potentiated by the PAMP-induced activation of the TLR cell-signaling cascade, as synthetized in Figure 1 , which would explain the fish improved ability to cope with pathogenic challenges. Thus, it is advisable in nutritional dose-response evaluating phytogenics to evaluate changes in expression in TRP receptors as well as gene markers of the TAK1/MAPK/NF-kB signaling pathways in order to provide insight into their mode of action at mucosal level.

Another limitation that should be taken into account when testing phytogenics is that most plant-derived bioactive compounds are either volatile and/or susceptible to rapid degradation in the stomach where acid digestion takes place, with consequent low availability at the intestinal level or uncontrolled changes in the dose of administration. Hence, to overcome this limitation and minimize potential losses, controlled releasing techniques, such as encapsulation or other coating technologies, can be used to improve the proper delivery of phytogenics. This technology allows a prolonged absorption and local availability of the bioactive compounds along the gastrointestinal tract, ultimately increasing their beneficial impact upon the host (158, 159). Moreover, encapsulation protects phytogenics from environmental degradation, such as from light, temperature and/or pH variations, and eventually playing an important role in their palatability, masking the potential pungency associated to some compounds that otherwise can affect feed intake (158). It is important to highlight that most of the studies considered in this review did not take into consideration those aspects, administrating phytogenics as powder forms, hydroethanolic extracts or dissolved solutions without proper assessment of their potential biodegradation during feed storage or along the gastrointestinal tract. The overall limitations identified in most of the currently available studies assessing fish immunity leads to the stigmatization of phytogenics application, in which compounds with high pharmacological value are labeled under the “medicinal plants” or “herbalism” pseudoscience stigma, with disbelieving scientific evidence. This represents a major restriction for the development of effective phytogenics at commercial scale.

Overall, it is fundamental that the efforts made in the research for sustainable prophylactic tools to boost host’s immune condition, stress resistance and pathogenesis prevention will culminate on reliable administration strategies for the aquaculture sector. Among the most studied group of natural bioactive compounds, both terpenes and organosulfur compounds have been suggested to display antimicrobial, antioxidant, anti-inflammatory and immunomodulating activities, with the potential of improving fish mucosal barrier function and integrity. Although they comprise a promising group of phytogenics for aquafeeds, an urgent update in the academical approach and experimental methodologies are needed to elucidate their pharmacokinetics and mode of action in depth. Therefore, in the present review we propose important molecular signaling pathways and hypothesize their involvement on the dietary immunomodulation in fish by the selected phytogenics.

JF and JG-V data analysis and visualization. JF wrote the draft. All authors contributed to the article and approved the submitted version.

JF is employed by TECNOVIT-FARMFAES S.L. FER-L is a senior research associate of the Consorcio Tecnológico de Sanidad Acuicola, Ictio Biotechnologies S.A. (Chile).

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.