Although fish lack organized lymphoid structures in their gut, the gut wall contains a range of immune cells such as plasma cells, other lymphocytes, and antigen-presenting cells (APCs) (17, 29). The mucosal barrier maintains homeostasis in the organism through local immune responses to sustain tolerance with mutual and commensal bacteria (30). Differentiated epithelial cells called goblet cells within the mucosa prevent pathogen entry by secreting mucins, high molecular weight glycoproteins that can contain antimicrobial properties. Goblet cells are a typical morphological marker to identify active mucosal immune responses, for example in brown trout (Salmo trutta) during parasitic infections, the secreted mucus contains acidic glycoconjugates that increase mucus viscosity for intestinal protection (31). Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) are lymphocytes that reside in the epithelial and lamina propria layers respectively, and recognize antigens to induce an adaptive immune response to maintain gut homeostasis and kill the pathogen (32).

The gastrointestinal tract of teleost fish has evolved to their aquatic lifestyle and feeding habits, but it is important to note that not all teleost fish have a stomach. For instance, zebrafish and common carp (Cyprinus carpio) lack stomachs and instead have an anterior intestine called the intestinal bulb, whereas fish like Nile tilapia (Oreochromis niloticus) and Atlantic cod (Gadus morhua) have a true stomach (3). The intestine is divided into three main regions: the pyloric caeca, midgut, and hindgut, which all aid in food digestion, nutrient absorption, and mucosal immunity. From their investigation in rainbow trout (Onchorhynchus mykiss) intestine, Verdile et al. (32) reported that gut segments should be accounted for when determining the relationship between metabolism and immune response. Typically, histological parameters used to determine gut health in response to dietary supplementation are based on classical morphological changes in the fish fed experimental and control diets, including villus length, lamina propria height, quantity of IELs, and qualitative changes in mucus secretion (32, 33). Differences in segment morphology could result from commensal bacteria populations within specific segments affecting the morphology of the brush border. Ultimately, these morphological changes can impact the expression of genes encoding immune signaling factors like chemokines and cytokines, which could lead to alterations in cellular signaling for immune defense. This is because enterocytes undergo rapid turnover to function properly and an imbalance between cell proliferation and apoptosis can lead to tissue dysfunction plus damage to the intestinal morphology (34).

Besides morphological analyses of the gut, other biomarkers present valuable information to better understand mechanisms impacted from supplementation. For example, intestinal alkaline phosphatase (ALP) activity is a biomarker for mature enterocytes and can be used in conjunction with the immunohistochemical localization of proliferating cell nuclear antigen (PCNA), where PCNA signal confirms the presence of stem cells (33). These signals tend to be within intestinal folds of the villi, and when identified with ALP activity, mature differentiated epithelial cells can be recognized (33). Alternatively, a lack of ALP leads to increased sensitivity to microbes and high levels of neutrophils, which modulate the composition and function of the gut microbiota (3). If pathogens have the ability to affect morphology as well as microbiota populations throughout the length of the gut, experiments should be cognizant of their impact on immune-relevant gene expression that alter mucosal defense (35). Published articles often either sample one gut segment or multiple segments, so validation is needed for past work utilizing only a single segment or less-defined ones to fully understand mucosal immunity throughout the gut length.

Production of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) depends on oxygen to fuel the mitochondria to meet the cell’s energy needs in an immune response (36). Circulating oxygen diffuses into tissues through the oxygen cascade gradient and functions as a regulator of ROS generation in the mitochondria (36). The mucosal barrier maintains homeostasis through the selective transport of nutrients including electrolytes and water from the intestinal lumen into the bloodstream while disposing of harmful substances to prevent them from entering the internal environment (34, 37, 38). Tight junctions, for example, can regulate the mucosal barrier by directly preventing toxins from entering the lumen. Development of the disease is closely associated with tight junction structure damage of intestinal epithelial cells that are triggered by pro-inflammatory cytokine released by the activation of the intestinal mucosal immune system (39–41). Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine that can disrupt the stability of tight junction, thereby introducing oxidative stress (41). Wu et al. (42), concluded from their work on Songpu mirror carp (Cyprinus carpio Songpu) that during Aeromonas hydrophila exposure, mRNA expression of tight junction structural protein genes was upregulated in intestinal mucosal cells to reduce intestinal permeability (42). This suggests that the regulation of mucosal tight junctions is to protect the integrity of the gut by controlling its environment and reduce oxidative damage. Intestinal epithelial cells communicate with the mucosa to facilitate an effective immune response, and this process starts with cellular signaling within the innate immune system.

Innate immune cells and related proteins play crucial roles to neutralize pathogens. The process begins with the recognition of pathogen-associated molecular patterns in fish gut (PAMPs) by pattern recognition receptors (PRRs) expressed on gut epithelial cells (13). This interaction detects and recognizes pathogens that disrupt homeostasis and initiates chemical, biological, and/or mechanical responses to prevent further infection (43). Toll-like receptors (TLRs) are a type of PRR that have been evolutionarily conserved to mediate innate immune defense, recognizing PAMPs such as lipopolysaccharides (LPS), a component of bacterial cell walls. In fish, LPS activates the expression of inflammatory cytokine genes, such as the interleukins, IL-1β, IL-8, IL-17A/F, and TNF-α, likely mediated by TLR4 (44). TLR-4 is an important receptor for inflammation because it activates the nuclear factor kappa B (NF-κB) pathway, leading to the release of inflammatory mediators, including chemokines and cytokines (42, 45, 46). Conversely, some fish do not present TLR-4 or an ortholog to recognize LPS (47, 48). A dual-luciferase reporter assay was used to compare NF-κB activation in gilthead seabream (Sparus aurata), spotted green pufferfish (Tetraodon nigroviridis), and zebrafish showed that LPS signaled a TLR4 independently from the MyD88 pathway (47). The MyD88 is a signaling protein for TLRs induced by NF-κB for immune signaling and interestingly the zebrafish TLR4 orthologs negatively regulated the MyD88-dependent pathway, suggesting that TLR4 is a negative regulator to provide resistance to bacterial endotoxins (47, 48). Other TLRs have been identified in teleost species such as rock cod (Eleginops maclovinus) (49). Different portions of the intestine were investigated following exposure to the bacterium Francisella noatunensis and modulated the transcription of innate PRRs TLR1, TLR5 and TLR8 were observed. The transcriptional profiles of the evaluated TLRs were likely based on the binding capabilities of TLR1 to bacterial lipopeptides and LPS, TLR5 to flagellin, and TLR8 to single-stranded bacterial RNA (49, 50). These findings showed that TLR1, TLR5, and TLR8 recognized F. noatunensis in different parts of the gut at different levels, further supporting that the receptors on the gut following infection modulate transcription differently amongst the length of the intestine (49). Activation of specific TLRs can cause downstream immune effects based on the cytokines and chemokines being produced.

Notable cytokines that can attract phagocytic cells to the site of infection include IL-1β, a proinflammatory cytokine produced by monocytes/macrophages that signals the proliferation of leukocytes and downstream production of additional chemokines and other cytokines (51). IL-2 is an example of a downstream cytokine that can further promote inflammation through increased signaling of IL- 1β and NO production to recruit neutrophils, while stimulating T cells of adaptive immunity (51, 52). IL-8 is a pro-inflammatory chemokine that induces neutrophil and macrophage migration during oxidative stress or when infected by a pathogen to clean cellular debris and generate new tissue (51, 53). IL-17A/F are cytotoxic cytokines that CD4⁺ helper T cells use to communicate inflammation through AMPs. IL-17A is produced by adaptive Th17 cells, inducing the expression of cytokines such as IL-1β, TNF-α, and IL-6 in yellow catfish (Pelteobagrus fulvidraco) (54). IL-17F is responsible for protecting epithelial cells against bacterial invasion, upregulating other proinflammatory cytokines such as TNF-α, a multifunctional proinflammatory cytokine that drives the proliferation of immunocytes and phagocytic activity. Hoseinifar et al. (51) reported that TNF-α could enhance the phagocytic ability, ROS activity, and apoptosis of granulocytes/monocytes, suggesting that the leukocyte phagocytosis mechanism could be regulated by TNF-α in rainbow trout (Oncorhynchus mykiss). Research in isolated leukocytes of largemouth bass (Micropterus salmoides) confirmed TNF-α’s role in phagocytosis, leading to an enhancement in phagocytic ability, ROS activity, and apoptosis of granulocytes (55).

Monocytes, macrophages, and neutrophils are granulocytes that mediate non-specific immunity by promoting inflammation and phagocytosis. Within macrophages, there are two major subpopulations: M1 and M2 macrophages. M1 has pro-inflammatory properties and expresses TNFα, whereas M2 has anti-inflammatory properties from increased expression of TGF-β, an anti-inflammatory cytokine (30, 56, 57). Monocytes and macrophages predominantly produce ROS and NO intracellularly, while neutrophils produce these molecules both intracellularly and extracellularly (4, 58–60). NO is a cytotoxic molecule of macrophages that mediates inflammation (26), where inflammation can be categorized as acute or chronic. Acute inflammation involves a series of events resulting in increases in blood flow as well as monocyte and neutrophil trafficking (61, 62). Acute inflammation can be shut down by inhibiting proinflammatory pathways and activating anti-inflammatory cytokines, like TGF-β. If the pathogen persists, the inflammation can become chronic by increasing blood flow through an increase in capillary permeability resulting from high amounts of antinutritional factors due to inadequate feed formulation (62–64). Chronic inflammation can lead to cellular resource depletion, tissue damage, and fibrosis unless it is optimally regulated by anti-inflammatory responses to neutralize ROS (63, 65). Acute inflammation can be prevented by inhibiting proinflammatory pathways and activating anti-inflammatory cytokines, such as TGF-β. If the pathogen persists, the inflammation can become chronic by increasing blood flow through an increase in capillary permeability resulting from either high amounts of antinutritional factors or inadequate feed intakes of nutrients (62, 63, 66). Chronic inflammation can lead to cellular resource depletion, tissue damage, and fibrosis unless it is optimally regulated by anti-inflammatory responses to neutralize ROS (63, 65). The cellular signaling in response to changes in an infection from acute to chronic depends on the cytokine and chemokines secreted from these immune cells. Inflammasomes provide surveillance, monitoring the infection site to promote the recruitment of immune cells causing inflammation, followed by expression of genes for anti-inflammatory signals to restore homeostasis and destroy damaged cells through an intracellular death process defined as apoptosis (67, 68).

In both fish and mammals, cytokines and chemokines are secreted through the bloodstream interacting with hepatocytes (68). This initiates crosstalk with various plasma proteins to signal for immune pathways like the complement system to be upregulated (69–71, 71, 72). The complement system in mammals includes three pathways with series of cascades of the cleaving until a terminal complex is lysed (69, 71, 73). Most subunits of the complement system have been identified in teleost fish and share structural similarities at the protein level with mammalian orthologs (69). Interestingly, teleost fish have variable isoforms of some components, notably C3 (74); C4 (75), C7 (76), C8 (77), C2/CFB (78), CFH (79), and CR3 (77). The various isoforms suggest different specificities for cleavage activation; however, the complement components are understudied, including the mechanisms involved to activate systems such as kallikrein (69, 80). Within the blood coagulation pathway in mammals, there is an important cascade with serine proteases called the kinin-kallikrein cascade that participates in blood clotting, blood pressure regulation and inflammation (81). Wong and Takei (82) found that the teleost fishes Nile tilapia and rainbow trout shared very little similarity in homologous structures of kininogens with mammals and had some components missing at the genomic level (82). A particular enzyme responsible for the creation of large structures with a high binding affinity had been lost through evolution within the teleost group (82). Instead, teleost fish have analogous structures within the kallikrein cascade and have a unique lectin activity known as the kalliklectin cascade (83). Andreeva et al. (81), investigated Atlantic cod serum for intracellular proteins to understand this cascade further, and observed no mammal-like osmotically active albumin but found osmotically active proteins that functionally replace the albumin independently from osmoregulation (81). The results from this experiment suggest that teleost albumin function was lost in fish during evolution, but other intracellular proteins are needed to maintain osmotic homeostasis (83). Their results demonstrated that these other plasma proteins are involved with transport in binding lectin to pathogens through the complement system and in immune regulation (83). To further explore the roles of neutralization of pathogens, we will discuss a crucial defense mechanism of teleost fish against invading pathogens: oxidative burst.

In teleost adaptive immunity, lymphocytes are activated by innate immunity or the antigen itself binding to the lymphocyte antigen receptor, producing a specified response to eliminate pathogens and affording memory protection (13). This activation begins at peripheral lymphoid tissues, including GALT, where naïve lymphocytes first encounter antigen to perform either cell-mediated immunity or humoral immunity (114). Cell-mediated immunity starts with naïve T cells, lymphocytes that have not been exposed to antigen that is specific for their antigen receptor. Naïve T cells will circulate through the blood and peripheral lymphoid tissues in search of the antigen of the pathogen, which will be presented by proteins of the major histocompatibility complex (MHC) on APCs such as dendritic cells (29, 114, 126). This specific antigen and co-stimulation will then activate the naïve T cells and spark proliferation and differentiation into effector cells such as cytotoxic T cells (CD8+) or helper T cells (CD4+), which will clonally expand and secrete cytokines and chemokines (29). Cytotoxic T-cells can target and kill infected cells via antigen presentation from MHC class I on the cell surface, where MHC class I high allelic polymorphism provides broad pathogen recognition at the host population level (114, 127). T-helper cells have diverse functions based on the cytokines or chemokines produced (114). For example, T-helper 1 cells (Th1) activate infected macrophage microbicidal functions via phagocytosis by antigen presentation by MHC class II. Th1 cells during an inflammatory response can secrete proinflammatory cytokines including IL-1β, TNFα and IFN-γ, that will activate M1 macrophages that will kill the pathogen (128). In contrast, during an anti-inflammatory processes, Th2 cells activate M2 macrophages that secrete cytokines IL-4 and IL-10 and synthesize polyamines to promote wound healing and defense against helminths (128, 129). Another noteworthy Th cell are Th17 cells that stimulate neutrophils for inflammatory responses, as well as maintaining homeostasis at the epithelial barriers (29, 114).

Teleost fish have B lymphocytes that produce immunoglobulins of three isotypes: IgM, IgD, and IgT/IgZ. Antibodies are immunoglobulins that are synthesized and secreted by plasma cells (30, 130–132). IgM are the primary immunoglobulin present and can also be found in the serum, while IgT/Z are mucosal-specific immunoglobulin analogous to mammalian IgA and is primarily found in the intestine and skin mucosal tissue (13, 114). Nomenclature was standardized to IgT from IgZ recently (133) although some teleost’s clades such as catfish lack the isotype signifying other potential mucosal immune defense mechanisms (134).

Lymphocytes proliferate with intense protein synthesis and, therefore, a sufficient bioavailability of AAs is important metabolic processes (including ATP production, oxidation, antioxidant defense, and the production of antibodies and cytokines) and immune responses (26). Protein synthesis requires a large amount of energy, with the minimum energetic cost estimated to be 40 to 50 mmol ATP equivalents per gram of protein synthesized (26, 135–137). In growing fish, about 20 to 30% of dietary AAs are used for protein accretion (138, 139). By combining knowledge from nutrition and immunology, we can develop immunostimulants through AA supplementation to prevent the damage caused by ROS, while still promoting effective immune responses when fish are infected. There has been extensive research done on mechanisms that regulate the antioxidant defense system in mammals but not with teleost fish. Some of the reasons are experimental limitations from the amount of serum or plasma in juvenile fish, as well as challenges to isolate the leukocytes of fish to measure ROS and NO production. It is critical to find solutions to using minimal amounts of samples to take these quantitative measurements to evaluate the health and welfare of these fish within aquaculture.

The nutritional requirements for AAs depend on numerous factors including the species, developmental stage, physiological status, gut microbiota, environment, and diseases (27, 140–143). Both immune response and antioxidant activity can be modulated through dietary supplementation as a non-invasive approach for controlling infectious diseases and minimize stress (21, 98, 144, 145). The teleost intestine has a plethora of digestive enzymes that facilitate the breakdown of complex nutrients into simple molecules that are absorbed into the intestinal mucosal cells by transporters. This process can regulate the downstream activation of the immune response through direct or indirect metabolic pathways. Understanding of their regulatory properties in the immune system in relation to AA requirements is imperative because protein feedstuffs contain different composition of AAs (138). For example, plant protein as the sole protein source in fish diets increases the risk of deficiency in important AAs like taurine, a sulfur-containing AA with antioxidant and osmoregulatory properties (138).

When using an AA as a supplement to aid immunity, it is important to understand its metabolic processes, including degradation in the gut, that will in turn affect the concentration of the AA in blood circulation, thus impacting dietary requirements (84, 98). To better understand this, we must evaluate digestibility and bioavailability of AAs prior to establishing an effective nutritional program for teleost fish. Digestibility of dietary protein is a percentage of AAs released from protein in the diet by the small intestine due to the actions of proteases and peptidases (84). This process is crucial because dietary proteins have no nutritional value unless hydrolyzed to free AAs and small peptides in the gut (84, 146, 147).

For fish, the dietary requirement for protein ranges from 30-60% of dietary dry matter, which is much greater than that for terrestrial mammals and birds (98). However the whole-body AA composition is largely similar among animals including fish, pigs, and chickens (138, 148, 149). For omnivorous teleost fish, their protein requirement is lower compared to carnivorous fish, and therefore the digestibility of plant-sourced feedstuffs is also important to consider because they generally have anti-nutritional factors including protease inhibitors (138, 150, 151). As mentioned previously, sustainable farming techniques are used in the aquaculture industry, including the use of plant-sourced feedstuff (such as soybean meal) instead of fish meal (84). In many regions of the world, the main plant-sourced ingredient is soybean meal; however, some teleost fish can have a limited capacity to use its protein because of a reduced activity of proteases in the digestive tract (53, 152). Important anti-nutrients present in soybean meal include not only protease inhibitors but also lectins, phytic acid, saponins, phytoestrogens, antivitamins, and allergens (150). There are two groups of protease inhibitors in soybean meal (trypsin inhibitors): Kunitz-type (153) and Bowman-Birk type 1 that reduces the digestibility of dietary protein (138, 154). These protease inhibitors can affect protein utilization by reducing trypsin activity in the small intestine by 30% (150, 155). To combat the effects of antinutritional factors, dietary supplementation with proteases can improve the digestibility of soybean meal in rainbow trout (138, 156).

The use of fishmeal has rapidly declined due to the serious environmental damages from high phosphorus content in fish fecal matter, creating poor water quality (157). Soybean meal is considered the most economic and environmentally friendly; however, compared to fishmeal, soybean meal is deficient in some AAs (e.g., glycine and methionine) and lacks taurine (158). Song et al. (157) performed a feeding trial in juvenile starry flounder (Platichthys stellatus) investigating differences in growth performance between diets with the protein component being fish meal or soybean hydrolysates (an improved soy protein with enzymatic modifications). They found that the soybean hydrolysates could replace part of fish meal in the diet without affecting the growth performance of the fish (157). This led to new initiatives of nutritionists within aquaculture to further understand protein metabolism in the context of fish health.

Intracellular protein undergo various cellular processes that regulates cellular integrity through protein synthesis and degradation (110). Proteases are separated by the membrane to control the rate of degradation during an immune response, like phagocytosis. Under stress, the lysosomal-vacuolar pathway is the dominant process for protein turnover in the liver, but other cells may rely on non-lysosomal proteolytic pathways (110, 159). Secretory factors from the duodenum, such as secretin produced by the S-cells, regulate secretions from the stomach, pancreas and liver, and cholecystokinin (CCK) by I-cells to stimulate the secretion of pancreatic digestive enzymes (84, 98, 147, 160). CCK aids in lipid digestion by first stimulating the gallbladder to contract and release the stored bile salts into the intestine, then signaling for enhancing the secretion of pancreatic digestive enzymes (84, 147). It is important to note that there are upper limits to dietary AA intake and that excessive AAs (e.g., > 2% glutamine and > 3% arginine in corn- and soybean meal-based diets for growing pigs) can become toxic (158). For example, supplementation with excessive methionine (e.g., 0.17% of diet) can reduce the life span in rats (158, 161). In addition, dietary supplementation with 563 ppm canavanine (a plant metabolite analogous to L-arginine) can reduce weight gain and feed intake in chicks (162). By contrast, dietary supplementation with 4% glutamate is safe for growing pigs (158).

Historically, AA requirements and functions in fish species have been determined primarily based on dose-response trials utilizing different protein sources. However, different feedstuffs have very different compositions of AAs (84, 147, 163, 164). Fatty acids and glucose can be stored in the body as triacylglycerols and glycogen, respectively, and when not utilized, they can accumulate and eventually impair the function of cells and tissues (138). Compared to mammals, fish liver and kidneys have high AA oxidation, but fish metabolism is more energetically efficient because they can excrete ammonia directly into their environment without needing ATP (26, 138, 141, 147). There are nine AAs that are not synthesized from any dietary AAs and have traditionally been regarded as nutritionally essential in all fish: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine (84). It remains unknown whether fish can synthesize arginine de novo (158). These AAs have a direct relationship with protein synthesis, so if one of the 9 nutritionally essential AAs is limited, it can impact intracellular protein synthesis and possibly proteolysis (26). Recently there have been suggestions to reevaluate the parameters for AAs because the term “non-essential” is a misnomer due to the critical role these AAs have in protein synthesis as well as immunity (84, 141, 147, 165). A widely accepted term, functional AAs, refer to AAs that not only participate in but also regulate key metabolic pathways. These AAs include arginine, glutamate, glutamine, glycine, and proline (138, 166, 167). Understanding optimal AA requirement in teleost fish cells is important for developing ideal diets and improving health through disease resistance, but only a handful of AAs have been extensively researched to investigates its health benefits. Table 1 summarize the current AA supplementation studies performed in teleost fish. We will discuss arginine as well as glutamate and glutamine in this review for they may directly influence antioxidant balance.