Evaluation of a commercial mushroom-derived product as a dietary immunomodulator and its effects on disease resistance in European seabass (Dicentrarchus labrax) juveniles

Highlights

• Dietary administration of Hifas Aqua at 1% and 2% modulated innate and adaptive immune-related pathways in European seabass.
• High inclusion levels upregulated inflammatory, antimicrobial and antigen-presentation genes after 110 days of feeding.
• High inclusion levels enhanced the humoral antimicrobial activity in the plasma.
• Fish fed 1% and 2% Hifas Aqua exhibited significantly higher survival following Photobacterium damselae subsp. piscicida challenge.
• These results support the use of mushroom-derived ingredients as sustainable immunomodulators in aquaculture.

1 Introduction

Global aquaculture reached a record production of 103 million tonnes in 2024, reinforcing its role as the main driver of growth in total aquatic animal production and accounting for 53% of the global aquatic animal production (FAO, 2026). However, the increasing incidence of infectious diseases and their associated environmental, social and economic impacts, particularly in intensive farming systems, represent a major constraint to the sustainable expansion of the aquaculture industry (Stentiford et al., 2017; Henriksson et al., 2021). The limited availability of effective prophylactic and therapeutic tools for controlling infectious diseases remains a major challenge (Pérez-Sánchez et al., 2018). Consequently, antibiotics continue to be widely used despite their association with the emergence of antimicrobial-resistant pathogens, immune system impairment, and adverse environmental impacts (Okeke et al., 2022). Because antibiotics are not effectively metabolized by fish, antimicrobial residues may accumulate in aquaculture systems, sediments and aquatic products, posing potential risks to environmental and human health. Moreover, excessive antimicrobial use may promote the selection of antimicrobial-resistant bacteria and facilitate the dissemination of antimicrobial-resistance genes through aquatic ecosystems and the food chain, contributing to the spread of antimicrobial resistance beyond the aquaculture environment (Santos and Ramos, 2018; Mushtaq et al., 2026). Therefore, there is an urgent need for sustainable alternative approaches capable of improving fish health and enhancing disease resistance.

In recent years, there has been growing interest in the identification and evaluation of bioactive compounds capable of modulating the immune response and enhancing disease resistance in farmed fish as sustainable alternatives to conventional disease management strategies. The incorporation of such compounds into aquafeeds represents a promising approach for improving fish health and preventing infectious diseases (Dawood et al., 2018). In this context, fungi have emerged as valuable sources of functional ingredients in aquaculture. Fungi comprise a highly diverse group of organisms, including molds, yeasts and mushrooms. Among them, mushrooms are macrofungi belonging to the phyla Ascomycota and Basidiomycota and have attracted considerable attention due to their high content of bioactive compounds, including dietary fibers, proteins and polysaccharides (Onomu and Okuthe, 2024). In particular, fungal cell walls contain pathogen-associated molecular patterns (PAMPs), such as glucans, which are major structural polysaccharides that have been widely reported to act as immunostimulants and health-promoting agents in several aquaculture species (Mensah et al., 2024). Medicinal mushrooms have been associated with improvements in innate immunity, disease resistance and health status across a range of aquatic species, although most studies have focused on freshwater animals and information in marine fish remains limited (Mohan et al., 2022).

Based on the immunomodulatory potential of fungal-derived compounds, Hifas Veterinary S.L. (https://www.hifasvet.com/I+D), the veterinary division of the biotechnology company Hifas da Terra S.L. (Pontevedra, Spain) has developed Hifas Aqua, a functional fungal-based product composed of a blend of mushroom species (Pleurotus ostreatus, Lentinula edodes, and Hericium erinaceum) enhanced with a proprietary strain known as Herimax^®. This product has been designed to improve fish health, support disease prevention and enhance productive performance in aquaculture. In this sense, dietary supplementation with H. erinaceum enhanced lysozyme activity, phagocytic activity, respiratory burst and improved resistance against Philasterides dicentrachi infection in Paralichthys olivaceus (Harikrishnan et al., 2011). Similarly, lentinan, a β-glucan isolated from L. edodes, modulated the expression of immune-related genes and altered inflammatory responses in rainbow trout (Oncorhynchus mykiss) (Djordjevic et al., 2009). However, studies evaluating combinations of medicinal mushroom species and their effects on immune-related gene expression and disease resistance remain limited, particularly in marine fish species. Furthermore, the immunomodulatory potential of Hifas Aqua has not yet been evaluated in European seabass (Dicentrarchus labrax). This species is one of the most economically important marine fish species cultured in the Mediterranean region, with aquaculture production reaching 262,017 tonnes in Europe in 2024, mainly driven by Turkey (160,000 tonnes), Greece (56,500 tonnes) and Spain (27,700 tonnes) (Federation of European Aquaculture Producers (FEAP), 2026). Furthermore, European seabass is considered a valuable animal model for studying fish immune responses because of the availability of immunological tools and extensive knowledge of its interactions with several bacterial, viral and parasitic pathogens (Picchietti et al., 2025). In fact, D. labrax is susceptible to numerous infectious diseases, including photobacteriosis caused by the Gram-negative bacterium Photobacterium damselae subsp. piscicida (Miccoli et al., 2024). This pathogen is associated with recurrent outbreaks, high mortality rates and significant economic losses in marine aquaculture, including European seabass farming (Rosado et al., 2019; Miranda et al., 2026).

The aim of this study was to evaluate the effects of dietary fortification with different inclusion levels of Hifas Aqua on immune response and disease resistance in European seabass juveniles. To this end, fish were fed either a basal diet (Control) or diets supplemented with 0.2% (HA0.2), 0.4% (HA0.4), 1% (HA1.0) and 2% (HA2.0) Hifas Aqua for 110 days. Immune status was assessed through the expression analyses of a panel of immune-related genes and the determination of lysozyme, peroxidase and bactericidal activities at different time points throughout the feeding trial and following a challenge with P. damselae subsp. piscicida. Moreover, disease resistance was evaluated through survival analysis following the bacterial challenge. Based on the previously reported immunomodulatory properties of mushroom-derived bioactive compounds, we hypothesized that dietary supplementation with Hifas Aqua would enhance immune responsiveness and increase resistance to P. damselae subsp. piscicida infection in European seabass.

2 Materials and methods

2.1 Experimental diets

Hifas Aqua (Hifas Veterinary S.L.) is a fungal-derived functional feed ingredient composed of P. ostreatus, L. edodes and H. erinaceus, including Herimax^®, a proprietary fermented H. erinaceus mycelium obtained from a patented strain (Patent No. P202230248). According to the manufacturer´s specifications, the product contains 164 mg g^-1 β-glucans (1,3-1,6) and 284 mg g^–1 α-glucans (1,3-1,6), determined using the Mushroom and Yeast Beta-Glucan Assay Procedure (Megazyme Ltd., Ireland).

Hifas Aqua (Batch No. 24032001) was incorporated into the experimental diets at different inclusion levels. The experimental feeds were formulated and manufactured by LifeBioencapsulation S.L. (Almería. Spain), a spin-off company of the University of Almería. The ingredients and proximate composition of the experimental diets are presented in Table 1. Five experimental groups were established according to the dietary inclusion level of Hifas Aqua: i) Control: fish were fed the experimental diet without Hifas Aqua supplementation; ii) HA0.2: fish were fed the experimental diet supplemented with 0.2% Hifas Aqua; iii) HA0.4: fish were fed the experimental diet supplemented with 0.4% Hifas Aqua; iv) HA1.0: fish were fed the experimental diet supplemented with 1% Hifas Aqua and v) HA2.0: fish were fed the experimental diet supplemented with 2% Hifas Aqua.

Table 1. Ingredient composition (% dry matter basis) and proximate composition (% as-fed basis) of the experimental diets used in the feeding trial.

Experimental diets were supplemented with Hifas Aqua (Hifas Veterinary) at inclusion levels of 0, 0.2, 0.4, 1.0 and 2.0%.

Control, HA0.2, HA0.4, HA1.0 and HA2.0 correspond to diets supplemented with 0, 0.2, 0.4, 1.0 and 2.0% Hifas Aqua, respectively. 1. Hifas Aqua is a fungal-based ingredient for animal nutrition composed of a combination of mushroom species (mycelium and body fruit blend). Composition: HERIMAX, fermented mycelium of Hericium erinaceus (Patent No. P202230248), Pleurotus ostreatus mushroom, Lentinula edodes mushroom, Hericium erinaceus mushroom. Composition: 12.56% crude protein, 2.40% crude fat, 6.60% crude fiber, 5.01% ash, 5.45% moisture, 164 mg g^-1 β-glucans (1,3-1,6), 284 mg g^-1 α-glucans (1,3-1,6).

2.2 Experimental design

The experimental design used to evaluate the effects of the inclusion of Hifas Aqua into the diet of D. labrax juveniles is shown in Figure 1. European seabass juveniles (average weight 15.83 ± 2.79 g), were supplied by Cupibar S.L. (Barbate, Cádiz, Spain) and transported to the facilities of IFAPA Centro El Toruño (El Puerto de Santa María, Spain). Following a 7-day acclimation period, fish were tagged using mini PIT (Passive Integrated Transponder) tags (Trovan^®, Dorset Identification B.V., Aalten, The Netherlands) and randomly allocated to five experimental groups (Control, HA0.2, HA0.4, HA1.0, and HA2.0) in triplicate, resulting in a total of 15 tanks connected to a closed recirculating aquaculture system (RAS). Diets were delivered using automatic feeders (Xclear Fish feeder ^©, Xclear, Schijndel, Netherlands) in 24 daily rations, distributed from 09:00 to 20:30. The amount of feed provided was adjusted weekly according to the estimated total biomass, calculated from previous sampling (2.5-1.5% of the total biomass). Throughout the experimental period, nitrite and ammonium concentrations were maintained below 0.1 mg l^–1, while water temperature, dissolved oxygen, and salinity averaged 20.80 ± 0.63 °C, 6.05 ± 1.62 mg l^–1, and 37.53 ± 1.49 ppt, respectively.

After 110 days post dietary treatment (dpt), juveniles (average weight 71.96 ± 3.77 g) were challenged with P. damselae subsp. piscicida (strain Lg41/01) supplied by Microbiology Laboratory of the University of Málaga (Andalusia, Spain) stored at -80°C in Tryptone Soy Broth (TSB; Cat. No. 413820.1210, PanReac AppliChem, Darmstadt, Germany) supplemented with 1.5% (w/v) Sodium chloride (NaCl; Cat. No. A2942,1000, PanReac AppliChem, Darmstadt, Germany) and 15% glycerol. P. damselae subsp. piscicida cells were grown on Tryptone Soy Agar (TSA; Cat. No. 413819.1210, PanReac AppliChem, Darmstadt, Germany) supplemented with 1.5% (w/v) NaCl at 22°C for 48 h and suspended in sterile phosphate buffered saline (PBS, pH 7.4) to 10⁹ colony forming units (CFU) ml^-1. Serial ten-fold dilutions were prepared and plated by microtitration on TSA supplemented with 1.5% (w/v) NaCl. CFU were counted after incubation at 22°C for 48 h to verify the concentration of the inoculum. A total of 250 juveniles were sedated with 2-phenoxyethanol (98%; Cat. No. 130240051, Thermo Scientific, Wilmington, DE, USA) at 200 ppm (Mylonas et al., 2005) prior to intraperitoneal injection. Fish were injected with 100 µl of the bacterial suspension containing 8.2 x 10⁸ CFU ml^-1 (8.2 x 10⁷ CFU fish^-1), corresponding to a mean dose of 1.14 x 10⁶ CFU g^-1 body weight, whereas control fish received 100 µl of phosphate buffered saline (PBS, pH 7.4) by the same route. This challenge dose was selected based on previous experimental infections of European seabass with P. damselae subsp. piscicida (Spinos et al., 2017). To minimize potential tank effects during the bacterial challenge, individually tagged fish were randomly redistributed into four tanks before injection. Two tanks were allocated to survival monitoring and two additional tanks to post-challenge sampling. Each tank contained 10 fish from each dietary treatment (Control, HA0.2, HA0.4, HA1.0 and HA2.0). Individual PIT tags enabled fish to be tracked and assigned to their corresponding dietary treatment throughout the challenge period. A total of 100 PBS-injected fish were maintained separately in two additional tanks under the same environmental conditions. Juveniles were not fed during the post challenge period and mortality as well as clinical signs of disease were monitored daily. Moribund fish were humanely euthanized and recorded as mortality events. The presence of P. damselae subsp. piscicida in spleen samples was assessed by multiplex PCR assay as described by (Amagliani et al., 2009), as well as bacteriological analysis using TSA supplemented with 1.5% (w/v) NaCl and incubated at 22°C for 48 h. At 2, 50 and 110 dpt, spleen and plasma samples were collected from three juveniles per replicate tank (n = 9 per treatment). Fish were anaesthetized with 2-phenoxyethanol (98%; Cat. No. 130240051, Thermo Scientific, Wilmington, DE, USA) at 200 ppm prior to sampling and subsequently euthanized by overdose of the same anaesthetic before tissue collection. At 2 days post challenge (2 dpc), spleen and plasma samples were collected from nine fish per dietary treatment randomly selected from the challenge tanks following the same anaesthesia and euthanasia procedures described above. Spleen samples were transferred to RNAlater™ Stabilization Solution (Cat. No. AM7021, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and stored at -80°C until use for gene expression analyses. Blood was drawn from the caudal vein and centrifuged at 6000 g for 10 min at 4°C. Plasma was collected and stored at -20°C until use for the determination of the lysozyme, peroxidase and bactericidal activity.

2.3 RNA isolation and RT-qPCR analyses

Total RNA isolation from spleen of European seabass juveniles as well as gene expression analyses through RT-qPCR were performed as described previously by Ponce et al. (2020) (Ponce et al., 2020a). Spleen samples were homogenized using the Lysing Matrix D (Cat. No. 6913-100, MP Biomedicals, Irvine, CA, USA) in a MiniG HG-400 homogenizer (Cole-Parmer, Vernon Hills, IL, USA) at 1500 strokes min^-1 for 2 min. Total RNA was isolated using RNeasy^® Mini Kit (Cat. No. 74106, Qiagen, Hilden, Germany) in accordance with the manufacturer´s protocol. Total RNA was treated twice with DNase I using RNase-Free DNase kit (Cat. No. 79256, Qiagen, Hilden, Germany) for 30 min. RNA sample quality was checked in agarose gels and quantification was accomplished using the NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Only RNA samples displaying intact RNA bands on agarose gels and A260/280 ratios between 1.8 and 2.1 were used for subsequent cDNA synthesis. Total RNA (1 μg) from each sample was reverse-transcribed using the iScript™ cDNA Synthesis kit (Cat. No. 1708891, Bio-Rad Laboratories, Hercules, CA, USA). Reverse transcription was performed at 25°C for 5 min, followed by 42°C for 30 min and 85°C for 5 min. Real-time PCR analysis was carried out on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Reactions were done in a 10 μL volume containing cDNA generated from 10 ng of original RNA template, 300 nM each of specific forward and reverse primers, and 5 μL of SsoAdvanced Universal SYBR^® Green Supermix (Cat. No. 1725272, Bio-Rad Laboratories, Hercules, CA, USA). A set of 10 genes related to the immune system was selected and RT-qPCR analyses were performed using specific primers (Table 2). Matching oligonucleotide primers were designed using Oligo v6.89 software (MedProbe AS, Oslo, Norway). The amplification protocol used was as follows: initial denaturation and enzyme activation for 30 s at 95°C, followed by 40 cycles of 95°C for 10 s, and 64°C for 30s. Each assay was done in duplicate. No-template controls (NTCs) and no-reverse transcription controls (No-RTs) were included in all runs to verify the absence of contamination and genomic DNA amplification. A melting curve analysis was performed at the end of each run to confirm amplification specificity. Data were normalized using the geometric mean of two reference genes: 40S ribosomal protein (rpsa) and elongation factor 1 alpha (eef1a). Reference gene stability was assessed using the CFX Maestro™ software (Bio-Rad Laboratories, Hercules, CA, USA), and rpsa and eef1a were identified as the most stable genes across all experimental groups and sample points. Relative mRNA expression was determined using the 2 (^-ΔΔCt) method (Livak and Schmittgen, 2001).

Table 2. Primers used for RT-qPCR gene expression analyses.

F and R refer to forward and reverse primers, respectively. The GenBank ID, the amplicon size and the efficiency of PCR reactions are indicated.

2.4 Lysozyme activity

Plasma lysozyme concentrations at 2, 50 and 110 dpt, as well as at 2 dpc, were determined using the Lysozyme Activity Kit (Cat. No. LY0100, Sigma-Aldrich, St. Louis, MO, USA). All samples were analyzed in triplicate using 96 well flat bottoms plates. Lysozyme from chicken egg white supplied with the kit was used for the construction of the standard curve ranging from 0 to 25 ng µl^-1. Micrococus lysodeikticus cell suspension was prepared at a concentration of 1.5 mg ml^-1 in the supplied reaction buffer and used as the substrate. Ten microliters of plasma or lysozyme standard solution were added into the wells and incubated with the substrate at 25°C. The decrease in absorbance was monitored at 450 nm every 5 min for 40 min using an Infinite^® M200 microplate reader (Tecan, Männedorf, Switzerland). The slope of the decrease in absorbance (ΔA450/min) was calculated for each standard concentration and used to construct the standard curve. Lysozyme concentrations in plasma samples were determined by interpolation of sample slope values against the standard curve. Samples were analysed on multiple microplates and concentrations were normalized to common reference samples to account for inter-plate variation.

2.5 Peroxidase activity

Total peroxidase activity in plasma was measured following the protocol described previously by Quade and Roth (1997) with slight modifications as reported by Ponce et al. (2024). Briefly, plasma samples were mixed with 3,3′,5,5′-tetramethylbenzidine (TMB; Cat. No. T2885, Sigma-Aldrich, St. Louis, MO, USA) and H₂O₂ (30%; Cat. No. H1009, Sigma-Aldrich, St. Louis, MO, USA). The reaction was stopped with 2 M sulfuric acid and the absorbance was measured at 450 nm using a microplate reader (Infinite^® M200; Tecan, Männedorf, Switzerland). One unit of peroxidase was defined as that which produces an absorbance change of 1 optical density.

2.6 Bactericidal activity

Plasma bactericidal activity was determined as described by Ponce et al. (2024). In brief, plasma samples were incubated with P. damselae subsp. piscicida (10⁶ CFU ml^-1) for 2.5 h at 25°C. Then, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Cat. No. M6494, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was added and the plate was incubated for 10 min at 25°C and centrifuged at 2000 g for 10 min. Finally, 200 µl of dimethyl sulfoxide (DMSO; Cat. No. D2438 Sigma-Aldrich, St. Louis, MO, USA) were added to each well and absorbance was measured at 560 nm. Percentage of viable bacteria was calculated as follows: 100 x (samples OD/positive controls OD). Bactericidal activity was calculated as the percentage of non-viable bacteria.

2.7 Statistical analyses

All data were tested for normality using a Kolmogorov-Smirnov test and for homogeneity of variance using Levene´s test. Data that did not comply with the assumptions of normality and/or homogeneity of variance were log-transformed prior to analysis. For gene expression analyses and lysozyme, peroxidase and bactericidal activity determinations performed at 2, 50 and 110 dpt, the mean value of the three fish samples from each tank was considered the experimental unit. A two-way analysis of variance (ANOVA) was conducted considering time and dietary Hifas Aqua treatment as fixed factors. When a significant interaction between sampling time and dietary treatment was detected, a one-way ANOVA was performed to compare treatments within each sampling point, followed by the post hoc Tukey test, with significance accepted at p < 0.05. Following bacterial challenge, PIT-tagged fish from all dietary treatments were redistributed among common challenge tanks. Therefore, analyses at 2 dpc were performed using individual fish as observational units and differences among dietary treatments were assessed using one-way ANOVA followed by Tukey´s post hoc test. Statistical significance was set at p < 0.05. Survival data were analysed using Kaplan-Meier survival curves and the log-rank test. Statistical tests were performed using IBM SPSS Statistics (version 22).

3 Results

3.1 Effects of dietary Hifas Aqua on D. labrax immune response during the feeding period

To evaluate the effect of dietary Hifas Aqua on the immune system response in European seabass juveniles, control groups were fed the basal diet, whereas HA0.2, HA0.4, HA1.0 and HA2.0 groups were fed the basal diet supplemented with 0.2, 0.4, 1.0 and 2.0% Hifas Aqua, respectively. Survival remained at 95% throughout the feeding trial, with no significant differences observed between the dietary treatments.

3.1.1 Immune-related gene expression

The expression profiles of a set of 10 genes related to the immune system were evaluated in the spleen of European seabass at 2, 50 and 110 dpt (Figure 2). Significant interactions between sampling time and dietary treatment were detected for all genes evaluated (two-way ANOVA, p < 0.05). When comparisons were performed within each sampling point, no significant differences were detected in the expression levels of any of the analyzed genes at 2 or 50 dpt (one-way ANOVA, p > 0.05). At 110 dpt, no significant differences were observed in fish fed the HA0.2 diet with respect to the control groups in any cases. In contrast, fish fed the HA1.0 and HA2.0 diets exhibited significantly higher mRNA levels for il1b, il10, tnfa, mhcI, mhcII, cd4, and cd8 mRNA levels compared with the control group (one-way ANOVA p < 0.05). Moreover, lyg expression was significantly upregulated in fish fed the HA2.0 diet, whereas significant increases in il6 and hamp expression were only detected in fish fed the HA1.0 diet (one-way ANOVA, p < 0.05).

3.1.2 Humoral innate immune parameters

A significant interaction between sampling time and dietary treatment was detected for all parameters analyzed (Figure 3, two-way ANOVA, p < 0.05). Lysozyme concentrations did not differ significantly among the treatments at 2 or 50 dpt. However, at 110 dpt, significantly higher lysozyme levels were detected in fish fed the HA0.4 and HA2.0 diets compared with the control group (one-way ANOVA, p < 0.05). Peroxidase activity was significantly higher in the HA1.0 and HA2.0 groups than in the control group at 2 dpt, whereas at 110 dpt, a significant increase was observed only in the HA2.0 group (one-way ANOVA, p < 0.05). Similarly, bactericidal activity did not differ among treatments at 2 or 50 dpt, but was significantly higher in fish fed all Hifas Aqua-supplemented diets compared with the control group at 110 dpt (one-way ANOVA, p < 0.05).

3.2 Effects of dietary Hifas Aqua following P. damselae subsp. piscicida challenge

3.2.1 Survival

At 110 dpt, European seabass juveniles were challenged with P. damselae subsp. piscicida (dose 10⁶ CFU g^-1) and survival was monitored for 9 days post challenge, dpc (Figure 4). Survival remained at 100% in juveniles from PBS injected groups throughout the experimental period. Mortality was mainly recorded between 2 and 6 dpc and progressively decreased with increasing dietary inclusion levels of Hifas Aqua. Pairwise Log rank comparisons revealed significantly higher survival rates in the HA1.0 and HA2.0 groups compared with the control groups (p < 0.05). Final survival rates were 6.7, 10.0, 16.7, 23.3, and 30.0% for the control, HA0.2, HA0.4, HA1.0, and HA2.0 groups, respectively. The corresponding relative percent survival (RPS) values were 3.6, 10.7, 17.9, and 25.0% for the HA0.2, HA0.4, HA1.0, and HA2.0 groups, respectively.

3.2.2 Immune-related gene expression

The expression profiles of the same set of 10 genes described above were assessed in the spleen of D. labrax juveniles at 2 dpc following challenge with P. damselae subsp. piscicida (Figure 5). Significant differences among dietary treatments were detected in the expression levels of il1b, il6, il10, mhcII and cd8 (one-way ANOVA, p < 0.05). The expression levels of il1b and il6 were significantly upregulated in fish fed the HA0.2 and HA0.4 diets, respectively, compared with the control. In contrast, il10 mRNA levels were significantly downregulated in fish fed the HA0.4 and HA1.0 diets. Expression levels of mhcII and cd8 were significantly higher in fish fed HA2.0 diet than in the control group.

3.2.3 Humoral innate immune parameters

Lysozyme concentration, peroxidase activity and bactericidal activity were determined in plasma of D. labrax juveniles at 2 dpc with P. damselae subsp. piscicida (Figure 6). Lysozyme concentration was significantly higher in fish fed the HA0.4, HA1.0 and HA2.0 diets than in the control and HA0.2 groups (one-way ANOVA, p < 0.05). Similarly, peroxidase and bactericidal activities were significantly higher in HA1.0 and HA2.0 groups than in the control, HA0.2, and HA0.4 groups (one-way ANOVA, p < 0.05).

4 Discussion

Disease prevention and effective health management have become essential components of sustainable aquaculture, as disease outbreaks remain among the most significant challenges facing the sector (Carlino-Costa and Belo, 2025). The use of bioactive compounds as functional feed ingredients represents a promising and sustainable strategy for improving fish health and enhancing disease resistance in the aquaculture industry. In this sense, medicinal mushrooms have attracted considerable attention due to their high content of bioactive molecules, including proteins, terpenoids, vitamins, minerals and polysaccharides such as chitin, α- and ß-glucans, mannans, xylans and galactans (Łysakowska et al., 2023; Sadowska et al., 2026). Products of medicinal mushrooms have been reported to increase both innate and adaptive immune responses, as well as disease resistance against pathogens in several aquatic species. Nevertheless, information regarding the efficacy of mushroom-derived functional ingredients in marine fish remains limited, particularly with respect to their effects on the expression profiles of immune-related genes (Mohan et al., 2022). The present study evaluated the effects of dietary fortification with Hifas Aqua, a fungal-based product composed of a combination of mushroom species (P. ostreatus, L. edodes and H. erinaceum), on immune response in European seabass. Inflammation constitutes a fundamental component of the immune response, playing a key role in host defense by promoting pathogen elimination and tissue repair. However, excessive inflammatory responses may be detrimental, causing tissue damage and impairing recovery processes. Dietary supplementation with Hifas Aqua induced a marked upregulation of the inflammatory cytokine-encoding genes il1b, il10 and tnfa at 110 dpt in the spleen of European seabass juveniles, particularly in fish fed the HA1.0 and HA2.0 diets. The simultaneous induction of both pro- and anti-inflammatory cytokines suggests a coordinated regulation of the inflammatory response aimed at enhancing immune protection while preventing excessive immune activation and tissue damage (Ouyang et al., 2011). These findings suggest that bioactive compounds present in Hifas Aqua were recognized by host pattern-recognition receptors, leading to the activation of immune signaling pathways and the subsequent induction of cytokine expression. The observed increase in lyg and hamp expression levels, together with enhanced plasma lysozyme, peroxidase, and bactericidal activities, is consistent with the activation of acute-phase and antimicrobial defense mechanisms associated with the innate immune response.

Lysozyme is one of the most important humoral defense factors in teleost fish, contributing to the degradation of bacterial cell walls and providing a first line of protection against invading pathogens (Magnadóttir, 2006). Similar increases in lysozyme activity have previously been reported in olive flounder (P. olivaceus) fed diets supplemented with Hericium erinaceus (Harikrishnan et al., 2011) and in rainbow trout (O. mykiss) fed diets containing L. edodes extract (Baba et al., 2015) and P. ostreatus extract (Uluköy et al., 2016). The increased plasma peroxidase activity observed in fish fed the highest inclusion levels of Hifas Aqua is consistent with previous studies reporting that lentinan extracted from L. edodes significantly enhanced peroxidase activity in the coelomocytes of the sea cucumber Apostichopus japonicus, indicating the activation of innate immune defense mechanisms (Wang et al., 2013). The increased bactericidal activity observed in the present study suggests an enhanced capacity of plasma humoral factors to inhibit or eliminate invading microorganisms. In this sense, polysaccharides isolated from Hericium caput-medusae enhanced bactericidal activity and disease resistance against Aeromonas hydrophila in grass carp (Ctenopharyngodon idella), supporting the role of mushroom-derived bioactive compounds in strengthening host antimicrobial defenses (Gou et al., 2018). These findings suggest that mushroom-derived bioactive compounds are able to enhance key humoral components of the innate immune system across different fish species, supporting the results obtained in the present study.

The stimulation of innate immune mechanisms observed in the present study was accompanied by changes in the expression of genes involved in the adaptive immune response. The adaptive immune response relies on antigen recognition and presentation mechanisms mediated by the major histocompatibility complex (MHC), which are essential for the activation and differentiation of T lymphocytes (Wilson, 2017). In the present study, dietary supplementation with Hifas Aqua induced a coordinated upregulation of mhcI, mhcII, cd4, and cd8 in the spleen of European seabass after 110 days of feeding with HA0.4, HA1.0, and HA2.0 diets, indicating that Hifas Aqua may enhance not only innate immune function but also immune mechanisms associated with the development of adaptive responses. To the best of our knowledge, studies evaluating the effects of medicinal mushroom-based feed additives on the expression of genes associated with antigen presentation and T cell activation in marine fish remain very limited. Although further studies are required to determine whether these transcriptional changes are associated with functional improvements in adaptive immunity, including antigen presentation, lymphocyte activation and pathogen-specific responses, the coordinated upregulation of antigen-presentation and T-cell-related markers strongly suggests an immunomodulatory effect of Hifas Aqua on adaptive immune pathways. Nevertheless, previous studies have reported enhanced humoral adaptive immune responses, including increased serum immunoglobulin levels, following dietary supplementation with medicinal mushrooms (reviewed by Mohan et al., 2022). These findings provide additional evidence that fungal-derived compounds may modulate adaptive immunity in aquatic animals through multiple mechanisms.

The immunomodulatory effects observed at both the innate and adaptive levels indicate a modulation of immune-related pathways in fish fed higher doses of Hifas Aqua. Although mortality remained high across all challenged groups, the increased survival observed in European seabass juveniles fed HA1.0 and HA2.0 diets indicates that dietary supplementation improved the ability of fish to cope with P. damselae subsp. piscicida infection under severe challenge conditions employed in this study. It is noteworthy that these dietary groups did not exhibit induction of inflammatory cytokine genes at 2 dpc. In particular, il1b and il6 expression levels remained comparable to those of the control groups, whereas il10 expression tended to decrease. The reduced expression of il10 in the most protected groups may further indicate a lower requirement for anti-inflammatory regulation, possibly reflecting more effective control of infection during the early stages of pathogen exposure (Ouyang et al., 2011). In this sense, a similar modulation of post challenge immune responses has been reported in rainbow trout fed lentinan, a β-glucan derived from Lentinula edodes. Following LPS stimulation, the supplemented fish exhibited a reduced induction of genes associated with acute inflammatory responses compared with the control fish, whereas other components of the immune response remained largely unaffected (Djordjevic et al., 2009). The authors suggested that such modulation may reflect a beneficial immunostimulatory effect, characterized by the attenuation of potentially detrimental inflammatory responses while maintaining effective immune defence mechanisms. In this study, fish fed HA1.0 and HA2.0 displayed significantly higher plasma lysozyme, peroxidase and bactericidal activities following infection. Furthermore, HA1.0 and HA2.0 dietary treatments promoted the upregulation of immune-related genes and enhancement of humoral defence activities before pathogen exposure. This pre-existing immunological status may have contributed to the differences observed after bacterial challenge. However, whether dietary supplementation induced a priming-like effect cannot be determined from the present data and would require additional analyses including pathogen-load measurements and longitudinal evaluation of immune responses. Similar priming-like responses have been reported in fish receiving immunostimulatory dietary supplements (Cabano et al., 2026). Collectively, these findings suggest that Hifas Aqua modulated innate and adaptive immune-related pathways before and after infection, and these immunomodulatory effects may have contributed to the higher survival rates observed following challenge with P. damselae subsp. piscicida.

While the present study provides evidence that dietary supplementation with Hifas Aqua modulates immune responses and improves survival following experimental challenge with P. damselae subsp. piscicida in European seabass, the biological effects observed cannot be attributed to any individual mushroom species or specific glucan component, as the product was evaluated as a complete commercial formulation. Directly comparable studies evaluating this specific mushroom formulation in European seabass are currently lacking, highlighting the need for further validation and replication studies. The study was conducted in a single life stage of European seabass using a single bacterial challenge model and a 9-day post-challenge follow-up period. Most of the immunological effects became evident only after 110 days of dietary supplementation, suggesting that prolonged administration may be required to achieve measurable immunomodulatory effects. Although HA1.0 and HA2.0 significantly improved survival following bacterial challenge, mortality remained high under the challenge conditions employed. Additional studies are needed to determine the optimal inclusion level and supplementation period and to assess the practical implications of long-term supplementation under commercial farming conditions. In addition, functional markers of adaptive immunity were not assessed, and conclusions regarding adaptive immune responses are therefore based primarily on transcriptional evidence. Future studies should investigate the molecular and cellular mechanisms underlying the observed immunomodulatory effects, including relevant receptor and signaling pathways, leukocyte function, complement activity, cytokine protein levels, antibody responses, bacterial load, histopathological alterations and pathogen clearance.

Overall, the results indicate that dietary supplementation enhanced the innate immune response and stimulated molecular pathways associated with adaptive immunity, as evidenced by the upregulation of immune-related genes and an increase in plasma immune activities after 110 days of feeding. Furthermore, fish fed the highest inclusion levels of Hifas Aqua exhibited improved survival following a challenge with P. damselae subsp. piscicida. Although the challenge resulted in high mortality in all experimental groups, the increased survival observed in HA1.0 and HA2.0 suggests that the immunomodulatory effects induced by dietary supplementation contributed to improved resistance to infection under the challenge conditions tested. These findings support the potential of mushroom-derived bioactive compounds as functional dietary ingredients for improving fish health in aquaculture and provide proof-of-concept evidence for the immunomodulatory effects of Hifas Aqua in European seabass.

Correction note

A correction has been made to this article. Details can be found at: 10.3389/fmars.2026.2023391.

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Footnotes

Edited by: Jorge García-Márquez, University of Porto, Portugal

Reviewed by: Tan Suet May Amelia, Chang Gung University, Taiwan

Ahmed M. Elsebaey, Mansoura University, Egypt