Fish skin mucus plays a distinct role in their defense mechanism. Mucus has powerful mechanisms that can trap and gather pathogens before they can contact the epithelial cells (Cone, 2009). Aquatic animals’ skin is a way for pathogens to enter the body. Therefore, such an evolutionary preparation was necessary (Fast et al., 2002). Currently, perhaps the most studied enzyme in fish mucus is lysozyme. Lysozyme bactericidal activity in fish cutaneous mucus and other tissues is considered a critical defense mechanism against pathogenic bacteria (Soltanian et al., 2018). In our results, there is no evidence that the mushroom powder has a remarkable effect compared to other additives. Shiry et al. (2019b; 2020) showed the administration of florfenicol (15 mg.kg− 1 BW for 10 consecutive days) leads to increased lysozyme activity, whether in serum or mucus, in rainbow trout without the challenge of experimental (healthy) infection. Previous studies indicate that feeding a diet containing A. bisporus powder leads to a significant increase in lysozyme enzyme activity (Safari et al., 2018; 2019). Comparing their findings with the present results indicating the presence of 1% WBMP in the diet can conduct to an increase in the activity of mucus lysozyme in the skin of zebrafish, but rainbow trout (1%), goldfish (1%), common carp (2%), and catfish (5%) require similar or different levels of WBMP in their diet, to observe the difference with the control groups (Khodadadian Zou et al., 2016; Safari et al., 2018; Shiry et al., 2020).
Other species of fungi such as oyster mushroom, Phellinus linteus, and Inonotus obliquus are renowned as immunostimulants in the aquaculture industry. The study of immunogenic effects on different fish, including Rainbow trout, brown trout (Salmo trutta), catfish (Silurus asotus), pangasius (Pangasius bocourti), longtooth grouper (Epinephelus bruneus), and olive flounder (Paralichthys olivaceus) confirms that these fungi can significantly increase the lysozyme activity against bacteria and parasites (Panigrahi et al., 2004; Harikrishnan et al., 2011; 2012a; 2012b; 2012c; Chang et al., 2013; Katya et al., 2014; Baba et al., 2015; Van doan et al., 2016). The immunomodulatory effects of WBMP (and powder or fungal extracts Lentinan and Schizophyllan) are probably due to the bioactive compounds of beta-glucan and other polysaccharides, which also approves the findings of Harikrishnan et al. (2012c), Chang and Wasser (2012), Przybylska et al. (2013), and Khodadadian Zou et al. (2016). In the present study, AGRIMOS was used as a positive control in supplementing the diet of a group of fish to compare the results obtained by white button mushroom powder.
Secretory immunoglobulins are mainly produced by plasma blasts and plasma cells and play a key role in providing mucus homeostasis. Examination of mucus samples obtained from various animal samples confirmed the presence of immunoglobulins (Harris & Hunt, 1973). Our results showed there was no significant difference in the amount of total immunoglobulin in the skin mucus between the experimental groups and the control group. Research by Lundén et al. (1999) and Reda et al. (2013) verifies the ineffectiveness of tilapia serum IgM and production of serum antibodies in rainbow trout, respectively. In the current study, no alternation in immune parameters was observed following florfenicol administration which is in contrast to the results of Shiry et al. (2019b;2020) narrated the suppressive effect of this oral antibiotic on rainbow trout. The difference between our results and the mentioned research may be due to the presence of experimental infection with streptococcosis/lactococcosis agents in that study, regulating the acquired immune behavior based on the loading of the pathogens in the blood. There are findings that the addition of WBMP alone or in combination with the probiotic Lactobacillus casei can lead to a significant increase in the level of mucosal total immunoglobulin in goldfish and zebrafish (Safari et al., 2018; 2019). Similarly, our results suggest that concomitant administration (antibiotic + WBMP) may have a synergistic effect on immunization, although this effect was not significant. Induction of mucosal Ig secretion following administration of 2% WBMP in the diet of common carp has been previously reported (Khodadadian Zou et al., 2016). In terms of the mechanism of the immune regulation effect, the presence of beta-glucan and other polysaccharides in fungi can be considered, which leads to stimulating the production and secretion of nitric oxide, cytokines, and prostaglandins in macrophages (Meena et al., 2013). It has been claimed that polysaccharides such as Lentinan and Schizophyllan can be stimulated by humoral immune parameters and T lymphocyte proliferation (Wasser & Weis, 1999).
Many studies have experimented with the effects of immune stimuli, such as plant stimuli, on the expression of lysozyme, TNF-α, and IL-1 genes (Nootash et al., 2013; Hoseinifar et al., 2015b; Safari et al., 2016) and, increased immunity in these fish has been reported through these additives. Fungi are employed as natural prebiotics and stimulate the immune response to make the beneficial populations of intestinal microbiota (Aida et al., 2009; Din et al., 2012; Van Doan et al., 2016). Assessment of lysozyme gene expression can be cited as a result of the effect of additives on this gene. The level of lysozyme or its activity is an important indicator in the primary defense of fish, and the level of activity of this enzyme or the level of mRNA expression changes in response to infectious diseases or stress. The most expression of the gene was related to the group that was separately taken the lower concentration (5 g per kg of FW) of WBMP, which are significantly different from other treatments. Increasing the activity level of this enzyme in the skin mucus of zebrafish was consistent with the same research of Safari et al. (2018; 2019). In addition, similar results were obtained for the effect of powder of the same fungus separately and in combination with L. casei by Safari et al. (2018) on increasing the expression level of lysozyme, which confirms our findings.
Tumor necrosis factor (TNF) is also a signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction. TNF-α plays a principal role in mediating proteins in immune cells and affecting immune cells (Gruss, 1996). It is also the chief mediator of the acute inflammatory response to gram-negative bacteria and other pathogenic microbes and is responsible for many systemic disorders of severe infections. This cytokine is a mediator between immune cells and serves as a biomarker and indicator of immune system activity in mammals and fish (Subramanian et al., 2007). Some antibiotics have a suppressive effect on increasing TNF-α (Er et al., 2010; Er & Yazar, 2012; Er & Dik, 2014). In the present study, the expression of the TNF-α gene decreased with increasing medicated feed doses in the groups that received florfenicol and mushroom powder both separately and in combination. In other words, this antimicrobial drug in high doses reduces the expression level of the gene associated with this inflammatory cytokine. These findings are matched with the results of other studies showing that TNF-α levels were increased in mice, rainbow trout (an infectious challenge), common carp (no challenge), rainbow trout (no challenge), goldfish (no challenge), and humans (Smiderle et al., 2011; Jeong et al., 2012; Khodadadian Zou et al., 2016; Harikrishnan et al., 2018; Shiry et al., 2019b; Safari et al., 2019). Contrarily, TNF-α levels in brown trout serum (LPS infectious challenge) remained unchanged (Ayse & Burak, 2014). Fucogalactans extracted from A. bisporus could also have an anti-inflammatory effect and reduced the level of cytokines such as TNF-α in the serum of mice (Ruthes et al., 2012). This may be due to the dose of antibiotic used, the route of administration or the dose of fungus used, or variations in animal species.
Cytokines are biological messenger proteins that play an immunizing role by affecting the function of macrophages and lymphocytes. This role is such that following the entry of external factors, genes involved in the development of inflammatory responses (tumor necrosis factor and interleukins) cause the concentration of macrophages and lymphocytes, which in turn leads to the inflammatory response (Zhou et al., 2002). Cytokine molecules can stimulate immune responses (Gruss, 1996). Gene expression of this inflammatory cytokine has also been induced following administration of antibiotics similar to the dose mentioned in rainbow trout challenged with infectious agents (Shiry et al., 2020). Compatible with these results, Khodadadian Zou et al. (2016) reported that white button mushroom powder (1 and 2% in the diet) had a significant effect on increasing the expression of the IL-1β gene in the intestines of common carp. One percent WBMP in rainbow trout diet also increased cytokine-related gene expression (Harikrishnan et al., 2018). The combination of WBMP (1%) with L. casei in zebrafish (Safari et al., 2018) and separately in common carp (Hoseinifar et al., 2018) increased the expression of the IL-1β gene. Given that the active ingredient in WBMP may be a beta-glucan derivative, Przybylska-Diaz et al. (2013) reported that fortification of the common carp diet with this immune stimulant for two weeks would increase IL-1β gene expression in the skin. The extract obtained from A. bisporus also induces IL-1β in human monocytes (Smiderle et al., 2011).
According to the results of the present study and other researches, it seems that the incorporation of 1% mushroom powder in the diet can improve some mucosal immune parameters in zebrafish. Apparently, the appropriate dosage of WBMP can act as an immune stimulant indicating dose-dependent property of this supplement which is similar to other beta-glucan resources. However, our recommendation is that given the positive effects that mushroom powder added to the diet alone, avoid taking antibiotics for this purpose. Hence, public health and environmental considerations can certainly be observed in this way.