Smenospongia aurea sponge samples (2.29 kg) were collected by SCUBA diving at a depth of 3.5 m in the Red Sea (from Hurghada with the aid of the Hurghada diving center GPS: N: 27.2574°, E: 33.8116°) in October 2023. The collected material was immediately frozen and kept at -20 °C until investigation and extraction. The sponge sample was identified by the National Institute of Oceanography and Fisheries (NIOF) in Alexandria. The extraction was performed in accordance with Esposito et al. (2015) as follows. The sponge material was thawed, chopped, homogenized, and then extracted as follows: a mixture of methanol (MeOH) and trichloromethane (CHCl₃) at a ratio of 1:1 was used for the extraction of Smenospongia aurea sponge samples at a solvent ratio of 1 w: 200 V while sited in a water bath shaker at room temperature for 48 h. The solvent, including the extract, was filtered using vacuum-boosted filtration and filter sheet Whatman No. 1 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). The solvent was separated from the filtrate by utilizing a high-capacity evaporator (EYELA-Rotary Vacuum Evaporator Apparatus) and then dried using a lyophilizer. The powdered extract was stored at 4°C until use. The bioactive components of the extract were analyzed using high-performance liquid chromatography (HPLC) (Agilent 1100, Merck KGaA, Darmstadt, Germany). The HPLC data revealed that the dominant active metabolites in the sponge extract derived from Smenospongia spp. were as follows: alkaloids (23.5%) such as imidazolidinones, dictazoline, and tubastrindole; terpenoids (46.43%) such as hydroquinone, quinone, ilimaquinone, sesquiterpenoids, smenoquinone, dactyloquinone, aureol, chloroaureol, smenodiol, sesquiterpene-aminoquinone, sesterterpenoids, tetronic acid, and cyclopentenone; chromene derivatives (3.75%) such as smenochromene; naphthoquinone (1.21%) derivatives such as smenocerone; and γ-pyrone polypropionate ( Supplementary Table S1 ).

Nile tilapia (Oreochromis niloticus) fingerlings were sourced from the Abbassa Fish Farm in Sharkia Province, Egypt. The experimental fish exhibited no notable superficial wounds. Prior to the experiment, 50 randomly selected tilapia fingerlings underwent a microbiological parasitological examination and proved to be free of F. columnare infection and Trichodina sp. infestation. The Nile tilapia were acclimated for 15 days (Jenkins et al., 2014) prior to the experiment initiation in an aerated dechlorinated fiberglass tank during which they were fed a control diet. Tank sediment, including fish feces and uneaten feed at the bottom of the fish tank, was eliminated daily, and clean, dechlorinated fresh water was used to replace approximately 25% of the tank water. All fish glass aquaria during the whole experimental period (80 cm length ×50 cm width × 70 cm height, filled with 90 L of dechlorinated water) were kept in the same culturing conditions where the dissolved oxygen was checked by an oxygen meter (Yellow Spring Instrument) and kept at 5.7 ± 0.3 mg/L, pH was checked via a pH meter (HANNA instruments) and kept at 7.39 ± 0.15, with temperature fluctuated between 23°C and 27°C. Total ammonia (0.42 ± 0.11 mg/L), nitrite (0.17 ± 0.17 mg/L), total hardness (9.32± 0.18 mg/L), and alkalinity (11.76 ± 0.08 mg/L, respectively) were evaluated three times per week via analytical kits (Alcon Ltda – Camboriú, SC, Brazil). The experimental protocols and exposure techniques were approved by the Institutional Fish Use and Care Committee of the Faculty of Veterinary Medicine, Zagazig University (ZUIACUC/2/F/114/2024).

A total of 625 healthy Nile tilapia fingerlings were randomly distributed into five experimental groups, each consisting of five replicates (N=125 fish/group; 25 fish/replicate), with an initial experimental body weight of 15.85 ± 1.8g, and each aquarium was considered a replicate. The experimental groups were as follows: the control group, which was fed a basal diet, and the other four groups which were fed the basal diet fortified with Smenospongia spp. extract (SS-extract) at doses of 50, 100, 150, and 200 mg/kg diet, respectively. For the determination of the initial doses of marine sponge extract before starting the experimental trial, approximately 100 fish were fed the basal diet with ten different concentrations of SS-extract (50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 mg/kg diet) for 21 days to optimize the starting concentration for the experimental groups and the supplemented groups exhibited no deaths, abnormal swimming behavior, skin lesions, or internal lesions. The fish were kept under daily observation during this period and all the observed fish showed no mortality, abnormal reflexes, or external lesions.

The nutrient composition of the experimental basal diet was designed based on the recommendations provided by the National Research Council (National Research Council, 1993) which ascertained the nutrient requirements of Nile tilapia, as indicated in Table 1 . The composition of the nutrients used in the feed formulation was confirmed using the standard analytical procedures (AOAC, 2002). The feed ingredients of the control diet were ground, sorted, and uniformly pelleted using a pelleting machine. Then, the SS-extract was added to the prepared feed in a consistent manner. The prepared diets were dried and then stored in preserving bags in cool (- 4°C) and dark places awaiting the start of the experiment. The fish were fed their prepared formulated experimental diets until apparent satiation three times per day at 07:00 am, 1:00 pm, and 07.00 pm for 60 days.

On day 60 of the experiment, growth performance-associated criteria were established as described by Ibrahim et al. (2021) as follows: at the start of the feeding trial, the initial weight of each fish in the respective tanks was recorded, and the average body weight along with feed intake (FI) was measured on a weekly basis. The growth performance of the Nile tilapia was calculated as ascertained by Alandiyjany et al., 2022; Eleraky et al., 2016; Ibrahim et al. (2021) as follows: Fish final body weight (BW) was assessed by individually weighing fish on day 60 of the feeding trial; Total feed intake (TFI) = total consumed feed for 60 days/fish number per glass aquaria; Total body weight gain (TFBWG, g) = body weight at the end of the experiment minus body weight at the beginning of the experiment; Weight gain % = TFBWG/average body weight at the outset of experiment ×100; feed conversion ratio (FCR) = Total feed intake for 60 days (g)/fish weight gain (g); Specific growth rate (SGR, %) = (LN body weight at the end of the experiment – LN body weight at the initiation of the experiment)/60 days ×100; Protein efficiency ratio (PER) = weight gain (g)/protein eaten (g); Fulton`s condition factor (K) = final fish weight (g)/fish length (cm)³ x 100; Survival rate = (fish count at the termination of the feeding trial/fish number at the start of the feeding trail) x 100.

To challenge fish with Trichodina sp, an experimental infection with Trichodina sp was done at the end of the feeding trial (60 days) for all the experimental groups as described by Obiekezie and Ekanem (1995). In total, 20 fish per group were randomly selected and transferred to 50 L glass aquaria and challenged with Trichodina sp by transferring 10 gill arches harboring 2,000 ciliates of Trichodina sp that were identified under an Olympus light microscope (Olympus, Tokyo, Japan) from diseased Nile tilapia obtained from the Fish Research Laboratory Unit, Faculty of Veterinary Medicine, Zagazig University. The fish were fed their corresponding diets throughout the parasitic challenge trial. All the infected fish were thoroughly monitored three times every day (8 am, 12 pm, and 5 pm) to record clinical signs and mortality (%) at 5, 10, 15, and 20 dpi (days post-infection). The infection rate (%) was evaluated as follows: (number of infected fish/number of fish sampled) × 100 at 5, 10, 15, and 20 dpi; and infection intensity was determined by preparing samples from two sites, gill filaments which were cut from both sides of each fish, and skin scrape samples using a glass coverslip (posterior margin of the caudal fin). Skin and gill samples were examined under an Olympus light microscope (Olympus, Tokyo, Japan) and parasite numbers were counted in a random manner by examining three viewing areas at 100X magnification (optic 10X and objective 10X, approximately 2.8 mm²), as described by Xu et al. (2015), for each sample at 5, 10, 15, and 20 dpi. Mucus secretion was scored as follows: (I) slight cloudy mucus on the side of the body, (II) one side of the body had cloudy mucus, (3) whole body had cloudy mucus, (4) the head and whole body had cloudy mucus, (5) head, nasal tip, and whole body had a thick layer of cloudy mucus.

To challenge the fish with F. columnare, isolates of F. columnare were previously obtained from diseased O. niloticus from the Fish Research Laboratory Unit at the Faculty of Veterinary Medicine, Zagazig University. The isolates of F. columnare were identified using the phenotypic and biochemical assays of Griffin (1992). The isolates of F. columnare were inoculated in modified Shieh broth and incubated aerobically for 24 h at 30°C. After 24 h of broth culture, the bacterial suspension was justified at an optical density (OD₅₉₅) of 0.3 via aseptic Shieh broth and tested for viability using the bacterial plate count method (in duplicate). After that, a ten-fold dilution of the bacterial suspension was spread on Shieh agar to an approximate colony forming unit (CFU) [3×10⁶ CFU mL−1 (OD₅₉₅ = 0.3)] and was then diluted to achieve an F. columnare challenge concentration by adding water in a 1:100 ratio into the tank to attain an LD₅₀ concentration of 3×10⁴ CFU/mL (Amphan et al., 2019) at 7 dpi. The fish were confirmed to be free from F. columnare preceding the challenge. In total, 10 fish per glass aquarium were randomly chosen to be challenged via the immersion method (10 L aerated water) with prepared bacterial broth at a concentration of 3 × 10⁴ CFU/mL for 2 h. Following the challenge, the fish were taken out of the infected suspensions, placed back into their assigned tanks, and kept under regular husbandry conditions. The challenged fish were observed for 30 days to identify any signs of columnaris disease. To confirm columnaris as the cause of death, F. columnare was isolated from the dead fish.

Upon completion of the feeding trial (day 60) and after challenge (9 dpi), 10 fish were anesthetized using ethyl 3-aminobenzoate methanesulfonate (30 mg/L, product No. E10521, Sigma-Aldrich, GmbH, Germany) before sample collection. Blood samples were collected twice [at the end of the feeding trial (day 60) and post-challenge (8 dpi)] from the caudal veins (5 fish were collected from each replicate, n=25 fish per group). Blood was collected either with EDTA anticoagulant for hematological assessment or without EDTA for serum separation via a configuration of 5,000 rpm for 10 min, and stored at -20°C until the examination of the biochemical and immune biomarkers. Furthermore, at the end of the feeding trial (day 60) and 8 dpi, skin and gill tissue samples (n=5/group) were collected and homogenized (1:10 w/v) in a buffer of Tris-HCl and centrifuged for 10 min at 2000 ×g for collection of supernatants, which were stored at −80°C until further assessment of oxidative and antioxidant biomarkers. Furthermore, the scraping of skin mucus was done using an aseptic plastic spatula from head to tail and was collected in sterile Eppendorf flasks and kept at -20 until further peroxidase and lysozyme analysis.

For the molecular analysis, other skin and gill tissue samples from representative fish (n=5/group) were collected, rinsed with normal saline solution, and then transferred to −80°C liquid nitrogen. For the collection of skin tissue samples for the evaluation of the total antioxidant capacity, skin tissues were cut into small cubes of approximately 3 cm, and then homogenized and centrifugated for estimation of total antioxidant assays, including a free radical scavenging assay employing 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS), and a ferric reducing antioxidant potential (FRAP) assay (Biochrom Ltd., Waterbeach, Cambridge, United Kingdom).

White blood cell (WBC) and red blood cell (RBC) counts were assessed via a diluted Natt-Herrick solution and a Neubauer hemocytometer (Sigma-Aldrich, UK). For the evaluation of hemoglobin (Hb) concentration, the cyanmethemoglobin colorimetric method was utilized. Blood hematocrit (Ht) was assessed by collecting whole blood with EDTA using micro-hematocrit tubes and centrifuging for 10 min at 12000 rpm. Serum biochemical indices, including cortisol hormone, were estimated by adopting the methodology certified by Chen et al. (2016), kidney function (creatinine and urea), total protein, albumin, globulin, alanine, and liver function [aspartate transaminase (AST) and alanine transaminase (ALT)] were assessed using a semiautomated clinical chemistry analyzer (Spinreact Co., Santa Coloma, Spain) according to the manufacturer’s guidelines. The serum and skin mucus lysozyme activity was evaluated by a turbidimetric assay following a procedure modified by Jenkins et al. (2014). Collected serum (20 μL) was incubated with lyophilized Micrococcus lysodeikticus [0.03% (w/v), 200 μL, Sigma, US)] at 22°C in sodium phosphate buffer (0.05mM, pH 6.2). After that, the absorbance was assessed at 490 nm via a Microplate Reader (Biochrom Ltd., Waterbeach, Cambridge, United Kingdom). Lysozyme activity was expressed as a reduction in absorbance of 0.001/min. The activity of the alternative complement pathway (ACH50) was evaluated using rabbit red blood cells for hemolysis according to the approach described by Sunyer and Tort (1995). The levels of serum NO were examined using the colorimetric method outlined by Bryan et al. and Grisham et al (Bryan and Grisham, 2007; Grisham et al., 1996). C-reactive protein (CRP) was tested via latex nephelometry based on the interaction with phosphocholine (Drieghe et al., 2014). Serum myeloperoxidase (MPO) levels were measured using the method of Suzuki et al. (1983). Immunoglobulin T (IgT) and immunoglobulin M (IgM) were analyzed via ELISA (enzyme-linked immunosorbent assay) kits from Sigma Aldrich, USA. The levels of serum complement C3 (CC3) were tested via Fish Complement Component 3 ELISA Kit (MyBioSource, Inc., San Diego, CA, USA). The respiratory burst activity produced by leukocytes was investigated with a microliter plate with the aid of 0.3% nitroblue-tetrazolium solution (Secombes, 1990). The serum and skin mucus total peroxidase activity was measured corresponding to Lazado et al. (2016). Fish serum (10 μL) was added to 90 μL of Hank’s Balanced Salt Solution free of Ca²⁺ and Mg²⁺, tetramethylbenzidine hydrochloride (20 μL, TMB, Sigma-Aldrich, St. Louis, MO, USA), and 5mM H₂O₂ (Sigma-Aldrich, MO, USA), and then incubated in the dark for 2 min, after which 4M H₂SO₄ (35 μL) was added to terminate the reaction. The absorbance was read at an optical density of 450 nm via a microplate absorbance reader against blank. Serum anti-protease activity was analyzed as described by Christybapita et al. (2007), with a few modifications. Fish serum (10 μL) was preincubated with a standard solution of trypsin (20 μL, Sigma-Aldrich, MO, USA) at room temperature for 10 min and then 2mM sodium-benzoyl-DL-arginine-p-nitroanilide hydrochloride substrate (BAPNA, Sigma-Aldrich, MO, USA) was added and incubated for 25 min at 20°C, and to terminate the reaction, trichloroacetic acid 30% (v/v) (TCA, Sigma-Aldrich, MO, USA) was added. The absorbance was determined at 415 nm using a Microplate Reader (Biochrom Ltd., UK).

Total antioxidant capacity (T-AOC, A015-1-2) was assessed using corresponding kits from the Nanjing Jiancheng Bioengineering Institute, China, according to the company’s recommendations. Serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities were estimated using Sigma 19160, 219265, and CS0260 kits, respectively, according to the manufacturer’s guidelines. Lipid peroxidation was analyzed by approximating the malondialdehyde (MDA) concentration using a Sigma MAK085 kit. Reactive oxygen species (ROS) was determined via the oxidation method described by LeBel et al. (1992). The hydrogen peroxide levels were tested according to Alves et al. (2021). A ferric-reducing antioxidant power (FRAP) assay (Benzie and Strain, 1996) was carried out on skin tissue homogenates. Homogenization was done in potassium phosphate buffer and centrifuged, and the supernatant was collected. Then, 1 mL of the supernatant was added to 3 mL FRAP buffer containing 10 mM TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine) in 20 mM Fe₂Cl₃, and 40 mM HCl was then added to 300 mM acetate buffer. Immediately following mixing, absorbance was read at 593 nm. A calibration curve was generated using FeCl₂. The antioxidant capacity of the samples was then determined and reported as micromoles (μM) of F⁺ per gram of wet skin tissue.

A DPPH assay was conducted according to Jang et al. (2008). In brief, skin tissue samples were homogenized and centrifuged. The supernatant was collected and blended with the solution of DPPH radicals and ethanol and then incubated for 10 minutes in darkness. The measurement of absorbance was read at 517 nm. The scavenging capacity for DPPH radicals was measured as μM/g of wet tissue.

The production of ABTS+ radical cations was stimulated by combining 14 mM ABTS with an equivalent volume of 4.9 mM potassium persulfate according to the modified Trolox-equivalent antioxidant capacity (TEAC) method of Manjunath et al. (2011). The reaction was then incubated for approximately 15 hours in a dark place. Subsequently, 1 mL of ABTS+ solution was combined with 10 µL of skin tissue homogenate and properly mixed, and the absorbance was measured at 734 nm after 60 s.

Tissue samples from skin and gills were gathered from the fish to assess the expression of immune response-related genes interleukin (IL-1β, IL-8, and IL-10), tumor necrosis factor-alpha (TNF-α), toll-like receptor 2 (TLR2), major histocompatibility complex (MHCIIβ), autophagy correlated genes [atg5 and 12, microtubule allied proteins 1A/1B light chain (LC3), and Beclin-1 (BCLN-1)], mechanistic targets of rapamycin (mTOR), antimicrobial peptide-related genes [β-defensin 1, hepcidin, anterior gradient protein 2 homolog (agr2), epithelial tumor suppressor (elf3)], mucin-2 genes, and endoplasmic reticulum stress-related genes [protein kinase RNA-like ER kinase (perk), eukaryotic translation initiation factor 2-alpha (eif2α), and activating transcription factor 4 (atf4), and Janus kinase 1 (JAK1)]. The collected RNA was isolated from a defined sample using a QIAamp RNeasy Mini Kit (Qiagen, Germany), according to the manufacturer’s instructions. The clearness and quantity of the extracted RNA were assessed using a Nano-Drop^® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA). The reaction was first subjected to a phase of denaturation for 12 minutes at 95°C. Later, denaturation ended at 95°C for 20 seconds, which continued for 40 cycles. Afterward, annealing was run for 30 seconds at 55-60°C. Then, elongation was conducted for 30 seconds at 72°C, and in the final phase, melting curve testing was done. For reverse transcription, RNA samples were converted to cDNA using QuantiTect Reverse Transcription (Qiagen, Germany) as per the guidelines provided by the manufacturer. The amount of 1 μL of cDNA transcript was blended with SYBR Green PCR mix (12.5 μL, QuantiTect SYBR Green Kit) and 0.5 μL of each forward and reverse targeted primer for each selected gene. A real-time PCR machine (Stratagene, La Jolla, CA, USA) was used for all reactions, and these were done in triplicate. The levels of mRNA of the examined genes were normalized against β-actin and calculated via the Ct (2^–ΔΔCT) process (Livak and Schmittgen, 2001). The gene sequences of the recognized primers are given in Table 2 .

Genomic DNA isolated from gill tissue samples was extracted by utilizing a QIAamp DNA fast DNA stool kit (Qiagen, Hilden, Germany). The DNeasy tissue kit was used and eluted with a volume of nuclease-free water (Promega, Madison, WI) equal to 1 L of water per mg of tissue. The quality and yield of DNA were detected spectrophotometrically via a Nanodrop (Thermo-Fisher Scientific, MA, USA).

The qPCR was performed via RT-qPCR assays in triplicate on the MX3005P real-time PCR machine (Strata-gene Co., USA) following the manufacturer`s protocol. In this context, the chondroitin AC lyase gene of F. columnare (GenBank accession number AY912281) (Panangala et al., 2007) was used as a housekeeping gene for rT-PCR. The F. columnare-specific primer (forward 5-CCTGTACCTAATTGGGGAAAAGAGG-3 and reverse 5-GCGGTTATGCCTTGTTTATCATAGA-3_) was used. The quantitative PCR assay to measure F. columnare utilized a total reaction volume of 10 μl. This included 5 μl of SYBR Green qPCR Master Mix from Qiagen (Australia), 2 μl of sample DNA, and 0.5 μl each of the forward and reverse primers. The analysis of the RT-qPCR results was conducted using the Rotor-Gene Q2 plex instrument from Qiagen Inc. (Valencia, CA, USA). The amplification conditions for detecting F. columnare were set as follows: an initial step at 50°C for 2 minutes, followed by denaturation at 95°C for 2 minutes, and then 40 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. Standard calibration curves for qPCR were prepared using 10-fold serial dilutions of DNA extracted from pure MG cultures. The quantities of MG were determined based on the number of target genomic DNA copies, and the resulting data were expressed as log₁₀ CFU per gram of the collected samples.

Variations in the obtained data among experimental groups were established via one-way and two-way analysis of variance (ANOVA) in SPSS version 21 for Windows (SPSS, Inc., Chicago, IL, USA) to distinguish the synergistic infection of Trichodina spp and F. columnare in response to different concentrations of SS-extract and their interaction. Levene’s test was applied to analyze the homogeneity of the variables preceding the statistical analysis while the normality of the obtained findings was detected by employing Kolmogorov–Smirnov’s test. Tukey’s-B post hoc test was selected to perform the various comparisons among the groups. The results were set to attain statistical significance at a p-value of 0.05.

After the 60-day feeding trial, an improvement in Nile tilapia growth performance parameters was observed after dietary supplementation with SS-extract ( Table 3 ). Remarkably, the groups fed SS-extract¹⁵⁰ and SS-extract²⁰⁰ displayed the most elevated (p < 0.05) final body weight, total body weight gain, and body weight gain percentage. Furthermore, the highest (p < 0.05) SGR and PER values were found in the SS-extract¹⁵⁰ and SS-extract²⁰⁰ supplemented groups. Regarding FCR, groups fortified with SS-extract¹⁵⁰ and SS-extract²⁰⁰ utilized their feed more efficiently than the other groups, which was reflected by decreased FCR values. The highest significant values for the Fulton condition K factor were detected in the SS-extract¹⁰⁰, SS-extract¹⁵⁰, and SS-extract²⁰⁰ groups. Meanwhile, the fish fed SS-extract at different levels exhibited no significant (p < 0.05) differences among them, unlike the control group.

The hematological and serum biochemical mediators prior to (at day 60) and post-challenge (at 10 dpi for the parasitic challenge and 3 dpi for the bacterial challenge) are presented in Tables 4 and 5 .

In all experimental groups, it was found that concurrent parasitic and bacterial challenge significantly (p<0.05) elevated serum creatinine, uric acid, cortisol, AST, and ALT levels with a reduction in RBCs, Ht%, and Hb. Notably, post the concurrent challenge, the elevated (p<0.05) levels of serum creatinine, uric acid, cortisol, AST, and ALT in the control group were significantly reduced in the higher SS-extract supplemented groups. Moreover, the groups supplemented with SS-extract¹⁵⁰ and SS-extract²⁰⁰ showed the highest (p<0.05) globulin and total protein levels. Finally, the group supplemented with SS-Extract²⁰⁰ exhibited a lower (p<0.05) level of cortisol with elevated RBCs and Hb compared to the non-challenged control group.

Remarkably, the capacity of skin tissues to eradicate DPPH free radicals was significantly (p <0.001) enhanced with increasing levels SS-extract compared to the control group. In the same way, the ability of skin tissues to scavenge ABTS reached its peak in the group supplemented with 200 mg/kg of SS-extract. Of note, the ferric-reducing antioxidant power of skin tissues was significantly increased in a dose-dependent manner, as reflected by a prominent reduction of Fe₃ ⁺ to Fe₂ ⁺ in the groups fed with SS-extract ( Table 6 ).

Data concerning antioxidants and oxidation-related parameters in skin and gill tissues prior to (at day 60) and post-challenge (at 10 dpi for the parasitic challenge and 3 dpi for the bacterial challenge) are listed in Tables 7 and 8 .

Regarding the effect of various levels of SS-extract in the skin and gill tissues, increasing the level of SS-extract significantly (p < 0.05) reduced MDA, ROS, and H₂O₂ and increased the activity of antioxidant enzymes, including CAT, SOD, GSH-PX, and TAOC, while the most prominent effect (p < 0.05) was observed in the SS-extract²⁰⁰ enriched group. Concerning the main influence of the challenge, exposure to a parasitic challenge followed by a bacterial one significantly (p < 0.05) elevated the oxidative stress-related biomarkers (MDA, ROS, and H₂O₂) and triggered the activities of antioxidant enzymes in all the experimental groups.

The infection intensity and rate (%) and mucus score were analyzed in the skin tissues of challenged fish at 5, 10, 15, and 20 dpi ( Figure 1 ). Among all the challenged groups, the highest (p < 0.05) infection rate and intensity and the lowest (p < 0.05) mucus score were detected in the supplemented groups. Furthermore, the minimum (p < 0.05) cumulative infection rate at different intervals was prominently higher in the SS-extract²⁰⁰ enriched group. In addition, at both 15 and 20 dpi (2-3 parasites per viewing area vs. 12 -14 parasites per viewing area in control), the SS-extract²⁰⁰ enriched group showed the most reduced (p < 0.05) infection intensity following the parasitic challenge. Remarkably, the mucus score reached its peak at 10 dpi and then decreased gradually at 15 and 20 dpi in the SS-extract-supplemented groups. There was a significant decline noted across all groups subjected to challenges (p < 0.05) in the mucus score at 20 dpi, especially with SS-extract²⁰⁰ supplementation. In the routine examination of Nile tilapia challenged with Trichodina spp., the fish exhibited dark coloration, lack of appetite, dullness, and abnormal swimming motion with markedly eroded skin and gills.

The immunological parameters tested in the serum of Nile tilapia prior to (at day 60) and post challenge (at 10 dpi for the parasitic challenge and 3 dpi for the bacterial challenge) are listed in Table 9 . Furthermore, the levels of peroxidase and lysozyme activity in the skin mucus are depicted in Figure 2 . The levels of peroxidase and lysozyme activity in the serum and skin mucus were markedly (p < 0.05) boosted by dietary supplementation of SS-extract in a manner that differed with the dose. Moreover, the highest significant levels of complement C3, ACH₅₀, antitrypsin percent, IgM, IgT, respiratory burst, and MPO were found in the group fed SS-extract at the level of 200 mg/kg of diet. Contrary to increasing the dietary level of SS-extract, serum NO and CRP values (p < 0.05) diminished dose-dependently. The challenge effectively (p < 0.05) exaggerated the immune response of Nile tilapia, as reflected by the increase in the analyzed serum immune-mediated parameters compared with their levels before the challenge in all the experimental groups.

The transcriptional levels of cytokine-related genes (IL-β, IL-10, IL-8, and TNF-α), MHCII β, and TLR2 genes in skin and gill tissues following the challenge are described in Figure 3 .

The mRNA expression levels of IL-10 and TLR2 were upregulated (p<0.05) in the skin and gill tissues at all intervals, and the highest levels were expressed in the SS-extract¹⁵⁰ and SS-extract²⁰⁰ groups. Additionally, the transcriptional levels of IL-β, IL-8, TNF-α, and MHCII in both skin and gill tissues were markedly decreased (p<0.05) at 15 dpi in the SS-extract supplemented groups, especially SS-extract²⁰⁰, while achieving their maximum expression in the control group.

The transcriptional response of the endoplasmic stressor mediator genes was assessed by estimating the expression levels of atf4, JAK1, PERK, and eif2α as shown in Figure 4 . The highest (p<0.05) response was found in the challenged non-supplemented group, as evidenced by the significant increase in atf4, JAK1, PERK, and eif2α gene expression levels. However, the expression levels of such genes were prominently lowered (p<0.05) in the skin and gill tissues in the group that was supplemented with SS-extract²⁰⁰, especially at 15 dpi.

Figure 5 illustrates the mRNA expression of autophagy genes at 5, 10, and 15 dpi post challenge. Groups supplemented with SS-extract significantly (p<0.05) upregulated Atg5 and 12, LC3-II, and BCLN1 and inversely downregulated (p<0.05) the expression levels of mTOR at all intervals when compared with the challenged un-supplemented control group. Moreover, at 10 dpi, the expression of Atg5 and 12, LC3-II, and BCLN1 reached its peak in the skin and gill tissues of the SS-extract²⁰⁰ group.

The mRNA expression levels of β- defensin 1, mucin-2, hepcidin, agr2, and elf3 in skin tissues at 5, 10, and 15 dpi are illustrated in Figure 6 . Through all intervals, the highest expression levels of β- defensin 1, mucin-2, hepcidin, agr2, and elf3 were cleared (p<0.05) at 10 dpi in all SS-extract-supplemented groups. The group supplemented with SS-extract²⁰⁰ displayed the most prominent upregulation (p<0.05) levels of β- defensin 1, mucin-2, hepcidin, agr2, and elf3 at 5, 10 and 15 dpi compared with the control group.

At 5, 10, 15, and 20 dpi, the mortality percentages for all the challenged experimental groups are illustrated in Figure 7 . At 5 dpi, the fish with a diet supplemented with SS-extract²⁰⁰ exhibited a lower (p<0.05) mortality percentage when compared with the other challenged experimental groups (3% vs 23% in the non-challenged control group). Furthermore, the accumulative mortality post-concurrent F. columnare challenge at 20 dpi was obviously elevated (p<0.05) in the challenged supplemented control group (reaching up to 78%). In contrast, the group supplemented with SS-extract²⁰⁰ exhibited the lowest (p<0.05) post-concurrent F. columnare challenge at 20 dpi (reaching up to 10%).

As shown in Figure 8 , supplementing the fish diet with SS-extract resulted in a significant (p<0.05) reduction in the F. columnare count at 3 and 5 dpi compared to the non-challenged control group. Notably, at 5 dpi, the lowest population of F. columnare was detected in the group whose diet was supplemented with 200 mg/kg SS-extract.