In Vitro and In Vivo Evaluation of Chaetoceros sp. Immunomodulatory Effects in Red Hybrid Tilapia, Oreochromis spp.

Simple Summary

Bacterial infections caused by Streptococcus agalactiae are a major challenge in tilapia aquaculture, leading to high mortality and economic losses. This study examined the immunomodulatory potential of the microalga Chaetoceros sp. in red hybrid tilapia. In the first phase, in vitro assays using fish blood and immune cells demonstrated that Chaetoceros extract at an optimal concentration enhanced lysozyme activity, which helps destroy bacterial cell walls, and promoted lymphocyte proliferation, a key component of adaptive immunity. Based on these findings, an in vivo feeding trial was conducted in the second phase. Tilapia were fed diets supplemented with Chaetoceros for eight weeks, resulting in improved lysozyme activity, stronger respiratory burst responses, and increased lymphocyte activity. Notably, fish fed a 2% Chaetoceros diet showed higher survival following a bacterial challenge with Streptococcus agalactiae. These results highlight Chaetoceros sp. as a promising natural immunostimulant to strengthen disease resistance and reduce reliance on antibiotics in aquaculture.

Abstract

Diatoms are recognized as a valuable source of bioactive compounds that can stimulate the immune defense mechanisms of fish. This study aimed to assess the effects of Chaetoceros sp. in modulating the specific and non-specific immunity of red hybrid tilapia through in vitro functional assays, an in vivo feeding trial, and a bacterial challenge. The in vitro experiment (Phase One) examined the immune response of tilapia cells exposed to Chaetoceros sp. extract, while the in vivo experiment (Phase Two) evaluated the immune response following an 8-week dietary supplementation with Chaetoceros sp. powder. In Phase One, an 8 mg/mL concentration of Chaetoceros sp. extract demonstrated an overall enhancement in lysozyme activity and lymphocyte proliferation. In Phase Two, tilapia fed a diet containing 2% Chaetoceros sp. showed significantly improved lysozyme activity, while the 5% supplemented group exhibited a significant increase in lymphoproliferation activity (p < 0.05). Growth performance parameters were generally comparable among dietary groups, indicating that supplementation did not adversely affect growth. Notably, the 2% diet also enhanced fish survivability following a challenge with Streptococcus agalactiae. These findings highlight the immunomodulatory potential of the diatom Chaetoceros sp. as a functional feed additive for freshwater fish, particularly red hybrid tilapia, and suggest its positive impact on fish health management in aquaculture.

1. Introduction

The rapid expansion of the aquaculture industry has led to increased fish production in various settings. However, disease management remains a major challenge, posing a significant threat due to its potential for devastating economic consequences [1]. The high-density environments characteristic of intensive aquaculture create a breeding ground for pathogens, making disease outbreaks a constant and perturbing threat. The economic repercussions can be severe, leading to high mortality rates and, in some cases, complete stock losses if not managed promptly [2]. Historically, antibiotics were the primary solution for treating infectious diseases on farms. However, their efficacy is diminishing due to the emergence of antibiotic-resistant bacterial strains [3]. The overuse of antibiotics also raises concerns about residues in aquatic food products and the environment [3,4,5], as well as the potential impact on human health [6,7]. Consequently, the presence of disease and antimicrobial residues can lead to trade restrictions, impacting international market access and causing further economic repercussions [8,9,10].

The immune health of fish is crucial for protecting them against a plethora of disease-causing agents, thereby ensuring consistent production and the sustainability of the aquaculture sector. A robust immune system enables fish to fend off not only occasional infections but also a wide array of pathogens—viruses, bacteria, parasites, and fungi—that can compromise their well-being [11,12]. Recently, it has become evident that fish feed can not only provide essential nutrients but also play a pivotal role in boosting the immune response of fish [13,14]. This evolution in aquaculture nutrition has led to the development of functional feeds, which are designed to integrate health management into diet formulations. Generally, functional feeds incorporate substances derived from natural sources known for their health benefits. These include a variety of components such as microalgae [15,16,17,18], macroalgae [19,20,21], herbal plants and seeds [22,23,24,25], and beneficial bacteria [26,27,28].

Among these, microalgae are particularly known for their wealth of bioactive compounds that can substantially regulate and enhance the immune responses of aquaculture species against pathogenic microorganisms. The immunomodulatory effects of microalgae in fish have been extensively reviewed [29]. Diatoms, a group of eukaryotic microalgae, are abundantly available in aquatic habitats and represent a promising yet underutilized resource in aquaculture immunology. Despite their potential, studies on the immunomodulatory properties of diatoms in fish have been comparatively scarce compared to other microalgal groups such as green microalgae [30,31,32], filamentous microalgae [33,34], and blue-green microalgae [35,36].

The diatom Chaetoceros sp. is renowned for its rich variety of beneficial bioactive components, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), sulphated polysaccharides, β-1,3-glucan, and fucoxanthin [37,38,39]. Recent research has focused on the antioxidant capacities and production optimization of these compounds within Chaetoceros sp. [40,41,42,43,44]. However, the immunoregulatory effects of Chaetoceros sp. are comparatively under-explored. Existing scientific evidence has shown promising results, suggesting that the inclusion of Chaetoceros sp. can improve certain aspects of immunity in aquatic animals such as shrimp and clams [45,46,47]. For instance, dietary supplementation with Chaetoceros muelleri has been shown to enhance the expression of immune-related genes and improve survival in whiteleg shrimp (Penaeus vannamei) following a bacterial challenge [45]. Similarly, Chaetoceros calcitrans has demonstrated antibacterial activity against fish pathogens and improved hematological parameters in infected common carp [46]. These previous applications highlight the potential of Chaetoceros sp. as a valuable immunomodulatory agent in aquaculture. Therefore, dietary supplementation with Chaetoceros sp. may provide significant advantages for the immune system of fish.

Building on this, the current research intends to explore the potential of the diatom Chaetoceros sp. in boosting both specific and non-specific immune systems in red hybrid tilapia (Oreochromis spp.). The specific objectives of this study are to determine the specific and non-specific immune responses of red hybrid tilapia to Chaetoceros sp. extract in vitro and to assess the effects of dietary supplementation with Chaetoceros sp. powder in vivo. As disease resistance and growth are among the most important criteria for assessing the suitability of feed additive candidates [48], this study also evaluates the growth performance and survivability of red hybrid tilapia fed a Chaetoceros sp.-supplemented diet.

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals used in the following experiments were of analytical grade. Reagents purchased from Sigma-Aldrich (St. Louis, MO, USA) include phosphate-buffered saline (PBS), fetal bovine serum (FBS), Leibovitz’s L-15 Medium with L-glutamine, zymosan A from Saccharomyces cerevisiae, nitroblue tetrazolium (NBT), Escherichia coli O111:B4 lipopolysaccharide (LPS), Concanavalin A (Con A), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), and Micrococcus lysodeikticus ATCC 4698. Dulbecco’s Modified Eagle Medium (DMEM) was procured from Gibco™, Thermo Fisher Scientific (Waltham, MA, USA). FBS, L-15 medium, and DMEM were sterile-filtered through 0.22 μm PES syringe filters. Tricaine methanesulfonate (MS-222) was obtained from Syndel (Ferndale, WA, USA). Dimethyl sulfoxide (DMSO) was acquired from Vivantis Technologies Sdn. Bhd. (Shah Alam, Malaysia). Trypan blue was purchased from R&M Chemicals (London, UK), while potassium hydroxide and methanol were obtained from Merck (Darmstadt, Germany).

2.2. Fish Husbandry

Red hybrid tilapia, Oreochromis spp. (initial weight: 14 ± 0.8 g), were obtained from a local fish supplier in Balakong, Selangor, Malaysia. The fish were transported in transparent plastic bags filled with oxygenated water to the Aquaculture Research Centre (Puchong station), Department of Aquaculture, Faculty of Agriculture, UPM. All experimental and analytical procedures were conducted in compliance with the UPM Institutional Animal Care and Use Committee (IACUC/AUP-R080/2018). One hundred and thirty fish were kept in a 1-tonne fiberglass tank and acclimatized to the environmental conditions for three weeks prior to the feeding trial. During the acclimatization period, fish were fed a commercial diet (32% crude protein, 6% crude fat) twice a day to apparent satiation. Daily mortality was monitored, and any dead fish were immediately removed from the tank to prevent the spread of diseases. After acclimation, the fish were randomly distributed into 12 rectangular 100 L glass aquaria, with four replicate tanks per treatment group. The stocking density was 10 fish per tank, and each tank was supplied with continuous aeration. The following water quality parameters were monitored using a YSI Pro Plus multiparameter instrument (Yellow Springs Instrument, Yellow Springs, OH, USA) throughout the experiment: temperature at 27 ± 3 °C; pH at 7.2 ± 0.8; dissolved oxygen at 6.93 ± 0.6 mg/L; and a photoperiod of 12 h light:12 h dark. Water management consisted of both daily and weekly exchange regimes. A partial water exchange of 10–15% was performed daily, during which faeces and uneaten feed were removed by manual siphoning to prevent organic accumulation. In addition, a 50% water exchange was conducted once weekly as a supplementary measure to maintain stable water quality. After each exchange, clean, dechlorinated water was added to restore the tank volume uniformly across all aquaria. Water quality parameters were monitored regularly throughout the experiment and remained within acceptable ranges for red hybrid tilapia, confirming that the water exchange regime was adequate to sustain optimal rearing conditions.

2.3. Microalga Chaetoceros sp.

The microalga used throughout the project was freeze-dried Chaetoceros sp. procured from Proviron (Hemiksem, Belgium). The nutritional composition of the batch used in this experiment was provided by the supplier as follows: 25–35% protein, 8–15% total neutral fat, and 35–45% carbohydrate, with the remainder comprising ash (10–15%) and moisture (5–10%). These values represent the typical range for this product and were used for the purpose of diet formulation.

In addition to the direct use of freeze-dried powder for dietary formulation, a methanolic crude extract of Chaetoceros sp. was prepared for the in vitro assays following the method described in [37] with slight modifications. Briefly, 100 mg of freeze-dried Chaetoceros sp. powder was extracted with 50 mL of methanol in a 100 mL amber Schott bottle (DURAN^®, Wertheim, Germany). The mixture was vortexed for 60 s (Vortex Genie 2T, Scientific Industries, Bohemia, NY, USA), followed by sonication at high speed for 30 min at room temperature (PowerSonic 505, HwaShin Technology Co., Seoul, Republic of Korea). The extract was filtered through two layers of Whatman™ Grade 1 qualitative filter paper, and the residue was re-extracted with an additional 50 mL of fresh methanol under identical conditions. The combined filtrates were concentrated using a rotary evaporator (IKA™ RV 8 V, Shanghai, China) at 30 °C, followed by drying under a fume hood. The dried extract was stored in a light-protected glass vial at −20 °C until use.

2.4. Overview of Immune Assays

Figure 1 illustrates the summary overview for the following experiment.

Red hybrid tilapia (Oreochromis spp.) was sacrificed for the following immunoassays. The immunoassays that were being carried out were lysozyme assays (involving blood serum, spleen, and head kidney leucocytes), respiratory burst assays (spleen and head kidney leucocytes) and lymphocyte proliferation assays (spleen and head kidney leucocytes). The assays were separated into two stages, namely Phase One (pre-feeding trial immune functional assay screening) and Phase Two (post-feeding trial functional assay evaluation). In Phase One, serum and tissue samples were obtained from untreated tilapias (25 ± 0.5 g) to obtain sufficient cells for the experiment. The leucocytes and serum were exposed to increasing concentrations of Chaetoceros sp. methanolic extract (0–10 mg/mL) prior to assessment in the respective immunoassays. Phase Two involved fish organ harvesting upon dietary supplementation with Chaetoceros sp. for 8 weeks. All experiments were performed in four replicates except for the lymphocyte proliferation assays in Phase One, which were done in three replicates.

2.5. Determination of Chaetoceros sp. Powder Inclusion Level

The inclusion level of Chaetoceros sp. powder for the in vivo feeding trial was determined based on the results of the in vitro assays (Phase One). The Chaetoceros sp. methanolic extract at a concentration of 8 mg/mL showed an overall significant improvement in the immunoassays (i.e., lysozyme assay and lymphocyte proliferation assay) and was therefore selected for subsequent dosage calculation. Based on this concentration, the amount of methanolic extract used in the in vitro assays was quantified. The extract amount required to enhance immune responses was calculated according to the in vitro results and corresponded to the average extraction yield obtained from methanol extraction of Chaetoceros sp. biomass. The dosage of Chaetoceros sp. powder (expressed as a percentage) was then back-calculated and adjusted according to the experimental fish body weight and daily feed intake, allowing conversion to an equivalent inclusion level in the formulated diet. The final concentration was established as 2% Chaetoceros sp. powder in the feed.

To enhance industrial applicability and economic feasibility for tilapia production, whole Chaetoceros sp. powder was incorporated directly into the diet rather than solvent-derived extract. Considering that tilapia is a relatively low-market-value species, large-scale extraction procedures would substantially increase feed production costs and reduce translational potential for commercial aquaculture. Therefore, the in vivo feeding design was intentionally structured to reflect realistic industry conditions. Although solvent extraction may concentrate specific bioactive fractions, whole microalgal biomass contains immunomodulatory compounds such as β-glucans, sulfated polysaccharides, carotenoids, and polyunsaturated fatty acids that are expected to be released during gastrointestinal digestion. Thus, the use of intact biomass better represents practical feeding scenarios.

In addition, a higher inclusion level of 5% was tested to evaluate dose-dependent immune responses in red hybrid tilapia. This dosage was based on the optimal concentration of Isochrysis galbana used by the same research team in previous studies [49], which demonstrated significant immunomodulatory effects. Furthermore, the 5% concentration falls within the range of immune-enhancing effects reported in various aquatic animals fed Chaetoceros sp. and other microalgae [15,47,50].

2.6. Diet Formulation

Fish meal (72%), soybean meal, fish oil, wheat bran, mineral, and vitamin premix were purchased from Nutri Vet Livestock Sdn. Bhd. in Negeri Sembilan, Malaysia. Vegetable oil and corn starch were obtained from a local market in Serdang, Malaysia. The diet formulation is listed below (

Table 1. Diet formulation for red hybrid tilapia containing different levels of Chaetoceros sp.

Ingredients	Control	2% Chaetoceros sp.	5% Chaetoceros sp.
Fish meal (g)	140	140	140
Soybean meal (g)	420	420	420
Wheat bran (g)	150	150	150
Corn starch (g)	250	230	200
Vegetable oil (g)	15	15	15
Fish oil (g)	15	15	15
Chaetoceros sp. (g)	0	20	50
Vitamin premix (g)	5	5	5
Mineral premix (g)	5	5	5
Total (g)	1000	1000	1000

). Briefly, the dry ingredients were weighed and uniformly mixed in a 10 L electric mixer (Golden Bull Mixer B-10, Kuala Lumpur, Malaysia). Then, oils and distilled water were added slowly until a homogenous dough was formed. The dough was pelleted using a screw-type pelleting machine (Golden Avill, Guangzhou, China). Subsequently, the finished pellet was spread evenly on trays and dried under a hot-air oven (Memmert, Schwabach, Germany) at 37 °C overnight. Once the pellets had completely dried, it was sealed in an air-tight plastic bag to prevent the growth of mould and kept in a dry place at room temperature until use. The inclusion levels of Chaetoceros sp. evaluated in this study (2% and 5%) represent relatively small proportions of the overall diet formulation. At these low inclusion levels, the nutritional contribution of Chaetoceros sp. to the total diet is minimal. Corn starch, which functions as a carbohydrate source in the basal diet, was proportionally reduced to accommodate the inclusion of Chaetoceros sp. Although corn starch contributes to the carbohydrate and energy content of the diet, the limited level of substitution (2–5%) resulted in no significant differences in the proximate composition of the experimental diets, as confirmed by proximate analysis (

Table 2. Proximate composition of experimental diets (% dry matter basis).

Ingredients	Control	2% Chaetoceros sp.	5% Chaetoceros sp.
Crude Protein	35.42 ± 0.38	35.67 ± 0.41	36.15 ± 0.45
Crude Fat	6.83 ± 0.22	6.95 ± 0.25	7.18 ± 0.28
Crude Fiber	4.21 ± 0.18	4.35 ± 0.20	4.58 ± 0.23
Ash	8.56 ± 0.31	8.72 ± 0.33	9.05 ± 0.38
Moisture	9.15 ± 0.28	9.08 ± 0.26	8.95 ± 0.24
NFE (Nitrogen-Free Extract)	35.83 ± 0.52	35.23 ± 0.48	34.09 ± 0.55
Gross Energy (MJ/kg)	17.85 ± 0.15	17.92 ± 0.18	18.05 ± 0.21

Note: Values are presented as mean ± SE (n = 3). The proximate analysis confirms that the experimental diets were essentially isonitrogenous (protein content: 35.42–36.15%) and isoenergetic (gross energy: 17.85–18.05 MJ/kg). The minor variations in protein and fat content (<1% difference) are attributable to the small inclusion of Chaetoceros sp. and do not represent biologically significant differences. Statistical analysis (one-way ANOVA) revealed no significant differences among the three diets for crude protein (p = 0.412), crude fat (p = 0.523), or gross energy (p = 0.687).

). Such substitution strategies are commonly adopted in functional feed additive studies, as minor reductions in corn starch (20–50 g per 1000 g diet) do not substantially alter the overall nutritional profile of the diet. This approach ensures that any observed effects can be attributed primarily to the bioactive compounds present in Chaetoceros sp. rather than to variations in macronutrient composition. Feed formulation was adopted from [50], in which the diet supplementation of microalga was shown to improve the growth and immune responses of Nile tilapia.

The proximate composition of the three formulated diets was analyzed according to the standard methods of the Association of Official Analytical Chemists (AOAC). Crude protein was determined by the Kjeldahl method, crude fat by Soxhlet extraction, crude fiber by acid-detergent fiber method, ash by incineration in a muffle furnace at 550 °C for 6 h, and gross energy by bomb calorimetry. The proximate analysis confirmed that the three experimental diets were nutritionally comparable, with only minor variations in protein and fat content attributable to the Chaetoceros sp. inclusion. The results, presented in Table 2, demonstrate that the diets remained essentially isonitrogenous and isoenergetic, supporting the validity of the experimental design.

2.7. Feeding Trial

The experimental diet given to the fish in each tank was completely randomized. Fish were fed one of three experimental diets: the control group was fed the basal diet, while the other groups were fed a diet supplemented with either 2% or 5% Chaetoceros sp. The amount of feed provided daily was 4% of their body weight (BW), with feeding performed twice a day at 9:00 a.m. and 4:30 p.m. for 8 weeks. Uneaten feed was siphoned out manually 30 min after each feeding, dried, and weighed to estimate daily feed intake and feed conversion ratio (FCR). Dead fish were recorded and removed from the aquarium daily to minimize the spread of infectious diseases.

2.8. Fish Serum Preparation

To obtain blood serum from fish fed the different experimental diets, four fish from each group (one fish from each replicate tank) were randomly caught and anesthetized with 250 mg/L of MS-222 [51]. Once the fish showed no signs of motion, it was brought to the dissecting board. A minimum of 600 µL of blood was collected from the caudal vein of each fish using a 1 mL syringe attached to a 25G needle. The blood was left to coagulate on ice for 4 h and then centrifuged at 1000× g for 10 min. The serum, located in the upper part of the tube, was gently aliquoted into a sterile tube to avoid mixing with the pellet. It was then stored at −20 °C until use [52]. Tilapias that were not fed Chaetoceros sp. were used in the Phase One in vitro assays mentioned in Section 2.4, and the protocols were the same as previously mentioned.

2.9. Organ Sampling and Leucocytes Isolation

Subsequently, the tilapia was euthanized with an overdose of MS-222 (400 mg/L; [53]), after which the head kidneys and spleen were removed aseptically. Each organ was meshed through a 40 µm cell strainer (Corning Inc., Corning, NY, USA) and centrifuged at 500× g for 8 min.

To isolate a greater variety of leucocyte types, hypotonic lysis of erythrocytes was performed based on [54] with slight modifications. Briefly, the cell pellet was suspended in 1 mL of phosphate-buffered saline (PBS 1×), to which 9 mL of cold sterile distilled water was added. The tube was inverted gently for 30 s to allow uniform lysis of the erythrocytes present in the sample. Following that, 1 mL of PBS 10× was added to restore the osmotic pressure to isotonicity, mixed thoroughly by gently inverting it back and forth, and immediately centrifuged at 500× g for 8 min. The resulting cell pellet was washed twice with PBS 1× accompanied by centrifugation to remove cell debris. After discarding the supernatant, the viscous cell pellet was tapped gently and resuspended in the desired volume of freezing media containing DMEM, 10% DMSO, and 10% FBS for cryopreservation. The cells were stored at −80 °C until analyses. Cryopreservation was employed to standardize assay timing and enable batch-wise processing of in vitro experiments under identical analytical conditions. All treatment groups underwent identical freeze–thaw procedures. Post-thaw viability was assessed, and cell concentrations were adjusted prior to assays. Although freezing may influence certain cellular functions, relative comparisons across treatments remain valid.

2.10. Determination of Cell Viability

For reconstitution, the frozen cells were thawed and immediately centrifuged at 500× g for 8 min. The supernatant was removed, and the pellet with cells was resuspended in Leibovitz’s L-15 Medium with L-glutamine supplemented with 10% FBS. Cell counting was performed within 30 min after reconstitution. The viability of the cells was assessed through a trypan blue exclusion test with the aid of a hemocytometer. The hemocytometer filled with cell suspensions was viewed under a compound microscope with 400× magnification. Cells were counted using a tally counter, where dead cells were stained with trypan blue and live cells remained unstained. The number of viable cells was determined using the formula below. To obtain the desired concentration, the cell suspension was adjusted with L-15 medium (supplemented with 10% FBS) to a desired concentration prior to the immunoassays.

$$\mathrm{viable} \mathrm{cell} \mathrm{count} (\mathrm{cells}/\mathrm{mL})={\displaystyle \frac{\mathrm{number} \mathrm{of} \mathrm{live} \mathrm{cells} \times \mathrm{dilution} \mathrm{factor} \times {10}^4}{\mathrm{number} \mathrm{of} \mathrm{larger} \mathrm{corner} \mathrm{squares} \mathrm{counted}}}$$

(1)

2.11. Lysozyme Assay

Lysozyme activity (LA) was assessed in serum, spleen and head kidney using modified turbidimetric assays [55,56]. The substrate adopted was a 0.02% (w/v) suspension of Micrococcus lysodeikticus ATCC 4698 made up of 0.05 M of sodium phosphate-buffered solution (pH 6.2). In Phase One, samples (25 µL) were mixed at a 1:1 ratio with Chaetoceros sp. crude extract (0–10 mg/mL), which was then added to 150 µL of M. lysodeikticus suspension. The reduction in absorbance was measured at 530 nm at room temperature after 0.5 min and 30 min of reaction. Lysozyme activity was calculated based on the change in absorbance over time. Phase Two repeated the same steps by replacing the microalga crude extract with PBS 1×. The sera used were not diluted with PBS whereas the concentration of tissue suspensions was adjusted to 1 × 10⁷ cells/mL for the assay. The results were reported in units of lysozyme activity per millilitre of serum or spleen/head kidney cell suspension, with one unit corresponding to a reduction in absorbance of 0.001 min⁻¹.

2.12. Respiratory Burst Assay

The estimation of respiratory burst activity (RBA) of splenic/head kidney cells was determined by nitroblue tetrazolium (NBT) assay [57,58]. Briefly, the number of viable cells (1 × 10⁶ cells/mL) for each organ sample was adjusted. Phase One involved the mixing of cell suspension (100 μL) with 50 μL Chaetoceros crude extract (0–10 mg/mL) followed by the addition of zymosan suspension (1:1 volume ratio, dissolved in 5 mg/mL PBS), whereas in Phase Two, Chaetoceros crude extract was not added. The mixture was incubated for 30 min at room temperature, after which the zymosan was removed by centrifugation at 447× g for 5 min. Then, the cell pellet was washed three times with PBS 1×. Approximately 100 μL of NBT dye was added and allowed to react for 30 min at room temperature. The NBT solution was removed via centrifugation and 100 μL of 100% methanol was added to terminate the reaction. The resulting formazan crystals were dissolved in 120 μL of 2 M KOH and 140 μL of DMSO. The optical density was measured at 630 nm using a microplate reader (Thermo Fisher Scientific Inc., USA). For negative control, the same steps were repeated without the addition of zymosan. The RBA was obtained using the formula below.

$$\mathrm{RBA}=\mathrm{Stimulated} \mathrm{activity} \left(\mathrm{SA}\right)-\mathrm{Basal} \mathrm{activity} \left(\mathrm{BA}\right)$$

(2)

where

SA refers to RBA due to the stimulation with zymosan;

BA refers to RBA without the stimulation of zymosan.

2.13. Lymphocyte Proliferation Assay

Lymphocyte proliferation assay was conducted based on Shiau et al. (2015) [59] with slight modifications. Basal and mitogen (T-cell and B-cell mitogen)-induced lymphocyte proliferation was assessed. Single cell suspensions were adjusted to 5 × 10⁵ cells/mL for the spleen and anterior kidney, respectively. In Phase One, approximately 100 μL of suspension was incubated with 25 μL of Chaetoceros crude extract (4–10 mg/mL) and 25 μL of mitogen solution with a condition of 27 °C for 48 h. Cell suspensions cultured without Concanavalin A (Con A) and Escherichia coli O111:B4 lipopolysaccharide (LPS) represented the non-stimulated cells. Phase Two involved incubation of 100 μL of cell suspension, 25 μL PBS 1× and 25 μL of mitogen solution under the same condition. The mitogen solution contained one of the following mitogens: 50 μg/mL of LPS or 20 μg/mL Con A. Cell suspensions incubated with the medium alone and mitogen were regarded as the control group. After the incubation was completed, the cell proliferation was evaluated using colorimetric assay based on colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) method, as described in Carvalho et al. (2018) with slight modification [60]. Briefly, the MTT dye (5 mg/mL dissolved in PBS 1×) was added to the cell mixture and incubated for 4 h at 27 °C, followed by centrifugation at 447× g for 10 min. The supernatant was discarded and the formazan crystals were dissolved by adding 100 μL DMSO. The contents were thoroughly mixed and the optical density at 570 nm of the resulting suspension was measured after 10 min of incubation. The stimulation index (SI) was presented using the following equation:

$$\mathrm{SI} = {\displaystyle \frac{\mathrm{Absorbance} \mathrm{of} \mathrm{the} \mathrm{stimulated} \mathrm{cells}}{\mathrm{Absorbance} \mathrm{of} \mathrm{the} \mathrm{non}-\mathrm{stimulated} \mathrm{cells}}}$$

(3)

In this study, T lymphocyte and B lymphocyte responses were functionally identified through their differential proliferative responses to specific mitogens. Concanavalin A (Con A), a plant lectin derived from Canavalia ensiformis, was used as a T-cell mitogen, as it selectively stimulates T lymphocyte proliferation by binding to glycoproteins on the T-cell surface [61,62]. Lipopolysaccharide (LPS) was used as a polyclonal mitogen commonly applied in teleost lymphoproliferation assays. In teleost fish, LPS responses are not mediated through the canonical mammalian TLR4 pathway [63,64]; therefore, the assay is interpreted as a functional leucocyte responsiveness test rather than a receptor-specific readout. This mitogen-based approach for distinguishing T and B lymphocyte responses is a well-established and widely accepted method in fish immunology studies [59,65,66]. The differential response to these mitogens provides a functional assessment of the adaptive immune response, reflecting the capacity of T and B cells to proliferate upon antigenic stimulation.

2.14. Assessment of Fish Growth Performance

At the end of the feeding trial, all fish were fasted for 24 h and weighed. The fish body weight and length of 30 red hybrid tilapia (from each experimental group) were measured, which was followed by dissection to weigh the livers. The following parameters were calculated based on the formula stated below: weight gain (WG), absolute weight gain (AWG), specific growth rate (SGR), hepatosomatic index (HSI), feed conversion ratio (FCR), and survival rate (SR).

$$\mathrm{WG} (\%) ={\displaystyle \frac{{(\mathrm{W}}_{\mathrm{F}}-{\mathrm{W}}_{\mathrm{I}})}{{\mathrm{W}}_{\mathrm{I}}}} \times 100$$

(4)

$$\mathrm{AWG} \left(\mathrm{g}\right) ={\mathrm{W}}_{\mathrm{F}}-{\mathrm{W}}_{\mathrm{I}}$$

(5)

$$\mathrm{SGR} (\%/\mathrm{day}) ={\displaystyle \frac{\mathrm{Ln} {\mathrm{W}}_{\mathrm{F}}-\mathrm{Ln} {\mathrm{W}}_{\mathrm{I}}}{\mathrm{T}}} \times 100$$

(6)

$$\mathrm{HSI} (\%) ={\displaystyle \frac{\mathrm{Liver} \mathrm{weight}}{{\mathrm{W}}_{\mathrm{F}}}} \times 100$$

(7)

$$\mathrm{FCR} (\%) ={\displaystyle \frac{\mathrm{FI}}{\mathrm{AWG}}} \times 100$$

(8)

$$\mathrm{SR} (\%) ={\displaystyle \frac{{ \mathrm{N}}_{\mathrm{F} }}{{\mathrm{N}}_{\mathrm{I}}}} \times 100$$

(9)

where W_F = final body wet weight (g); W_I = initial body wet weight (g); T = experimental period (days); N_F = final number of fish; N_I = initial number of fish; and FI = fish feed intake (g).

2.15. Bacteria Culture and Determination of Viability

The pathogen used in this challenge was Streptococcus agalactiae serotype III which had been characterised extensively in a previous article [67]. It was originally isolated from infected tilapia in a pond with disease outbreak. S. agalactiae strain was cultured using Brain Heart Infusion (BHI) at 37 °C, and subculture was performed daily before it was used. For each subculture, one loopful of the culture was streaked on BHI agar and incubated overnight at 37 °C to confirm the purity of the pathogen. Concentration and viability of S. agalactiae were determined by performing spread plate method further confirmed with microscopic cell counting via haemocytometer.

Spread plate method is briefly described below. The overnight broth culture was aliquoted into several tubes, and the aliquots were diluted into respective concentrations (ten-fold dilution series) using suitable diluent. Approximately 0.1 mL of diluted suspension was spread evenly on the agar and incubated overnight at 37 °C. The colony forming units (CFUs) with a range of 30–300 CFUs were employed to calculate the CFUs/mL of original sample, using the below formula:

$$\mathrm{Concentration}, \mathrm{CFU}/\mathrm{mL} = {\displaystyle \frac{\mathrm{number} \mathrm{of} \mathrm{colonies} \mathrm{counted} \times \mathrm{dilution} \mathrm{factor}}{\mathrm{volume} \mathrm{plated}}}$$

(10)

Furthermore, a trypan blue exclusion test was employed. To begin with, the broth was removed from the bacterial culture via centrifugation at 800× g for 10 min and the pellet with cells was resuspended in phosphate-buffered saline (PBS). Cell counting was performed within 30 min after reconstitution using 0.2% trypan blue solution. The haemocytometer was filled with cell suspensions and was viewed under a compound microscope with 400× magnification. Cells were counted using a tally counter, where the dead cells were stained with the colour of trypan blue and the live cells remained unstained. The number of viable cells was determined using the formula below:

$$\mathrm{viable} \mathrm{cell} \mathrm{count} (\mathrm{cells}/\mathrm{mL}) = {\displaystyle \frac{\mathrm{number} \mathrm{of} \mathrm{live} \mathrm{cells} \times \mathrm{dilution} \mathrm{factor} \times {10}^4}{\mathrm{number} \mathrm{of} \mathrm{larger} \mathrm{corner} \mathrm{squares} \mathrm{counted}}}$$

(11)

2.16. LC₅₀ Determination

Before the implementation of the challenge test, a preliminary investigation was conducted to obtain the mean lethal concentration (LC₅₀) of S. agalactiae in 14 days. The fish used for the LC₅₀ determination were from the same batch as the main experiment and were pre-screened for common tilapia pathogens (e.g., TiLV, Aeromonas hydrophila) to ensure they were disease-free. The LC₅₀ was determined via bath immersion of S. agalactiae at room temperature, utilizing concentrations of 1 × 10⁸, 10⁹, 10¹⁰, 10¹¹ CFU/mL. Untreated tilapias procured from the same supplier were subjected to a two-week acclimatization period, during which they were fed a commercial diet, and any dead individuals were immediately removed from the aquarium to minimize potential disease transmission risks. Following that, the fish were randomly divided into four groups, each in triplicate, with 10 fish per tank. Each group was challenged with their assigned bacteria concentration. Cumulative mortalities were observed and recorded on a daily basis, followed by further confirmation of any signs or symptoms suggestive of Streptococcal infection. Symptoms of fish infected with Streptococcus sp. included exophthalmia (pop-eye), reddened eyes, opaque corneas, splenomegaly, pale gills, and the presence of ulcerations [68]. The concentration of S. agalactiae that resulted in 50% lethality of the tilapia was determined for the following experimental assessment.

2.17. Bacteria Challenge Test

Streptococcus agalactiae was cultivated one day prior to the challenge test, adhering to the protocols stated in Section 2.16. The bacteria culture was centrifuged at 800× g for 10 min. The supernatant was discarded, and the resulting cell pellet was washed three times with phosphate-buffered saline (PBS 1×) to remove cell debris. Thereafter, the suspension was adjusted to the desired density of S. agalactiae through the addition of PBS 1×. The challenge test was carried out in triplicate, and we adopted the fish that had been subjected to the feeding trial (i.e., 10 fish per tank, a total of 90 fish). The fish were challenged via intraperitoneal (IP) injection with a standardized dose of S. agalactiae (1 × 10⁷ CFU/fish). The infected tilapias were monitored for a period of 14 days to assess mortality. Mortality was recorded daily, and the cumulative mortality was calculated. The survival rate was determined at the end of the 14 days.

2.18. Statistical Analysis

All data were analyzed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). The results are presented as mean ± standard error (SE). Before analysis, all data were tested for normality using the Shapiro–Wilk test and for homogeneity of variances using Levene’s test. For data that met the assumptions of normality and homogeneity of variance, a one-way analysis of variance (ANOVA) was used, followed by Tukey’s honestly significant difference (HSD) post hoc test for pairwise comparisons. For data that violated the assumption of homogeneity of variance, Welch’s ANOVA was used, followed by the Games–Howell post hoc test. Survival data from the bacterial challenge were analyzed using Kaplan–Meier survival analysis with the log-rank test to compare survival curves among the treatment groups. A p-value of <0.05 was considered statistically significant.

3. Results

This section presents the findings from the research evaluating the immunomodulatory effects of Chaetoceros sp. on white blood cells and blood serum of red hybrid tilapia. The results are presented by functional assay, with each assay including both Phase One and Phase Two findings. Phase One involved isolating immune components such as leucocytes and serum, mixed with different concentrations of Chaetoceros sp. extract in vitro to study functional assays and effects; in Phase Two, fish were fed varying dosages of Chaetoceros sp. incorporated feed, and immune components were then isolated and analysed through functional assays.

3.1. Lysozyme Activity

This investigation aimed to determine the potential of Chaetoceros sp. extract in modulating the lysozyme activity of red hybrid tilapia at varying concentrations. Blood serum and leucocytes derived from the spleen and head kidney of red tilapia were adopted to assess the effects of different levels of Chaetoceros sp. extract. The resulting activities are illustrated in Figure 2, alongside the lysozyme activity upon supplementation of Chaetoceros sp.

In Phase One, the lysozyme activity in blood serum (Figure 2A) showed a significant increase at concentrations of 4, 8, and 10 mg/mL compared to the control group (p < 0.05). However, a significant decrease in lysozyme activity was observed at lower concentrations (0.8, 1, and 2 mg/mL). This biphasic response may be attributed to a hormetic effect, where low doses of a substance can have an inhibitory effect while higher doses have a stimulatory effect. In spleen leucocytes (Figure 2C), lysozyme activity was significantly enhanced at concentrations of 8 and 10 mg/mL. In contrast, the head kidney leucocytes (Figure 2E) did not show any significant variations across the different concentrations of Chaetoceros extract tested (p > 0.05).

In Phase Two, dietary supplementation with Chaetoceros sp. powder modulated lysozyme activities. Notably, the serum lysozyme activity of tilapia (Figure 2B) fed with 2% Chaetoceros sp. demonstrated a significant increase compared to both the control group and the 5% Chaetoceros sp.-supplemented group (p < 0.05). The lysozyme activities of spleen and head kidney (Figure 2D,F) in fish fed either a 2% or 5% Chaetoceros sp. diet showed a remarkable increase compared to the control group (p < 0.05). However, there was no significant difference between the 2% and 5% supplemented groups.

3.2. Respiratory Burst Activity

In addition, the potential of Chaetoceros sp. extract in regulating respiratory burst activity was investigated in red hybrid tilapia following the lysozyme assay evaluation. Figure 3 presents the assessment of the immunomodulatory effect of Chaetoceros sp. extract on tilapia spleen and head kidney leucocytes, covering both Phase One and Phase Two trial.

Among the cellular immune responses from Phase One, the respiratory burst activity showed subtle changes. No significant differences were observed in the respiratory burst activities of either the spleen or the head kidney across all concentrations of the microalgal extract being tested (p > 0.05) (Figure 3A,C).

Meanwhile, the observations of Phase Two strongly support the hypothesis that Chaetoceros sp. is able to modulate the immune systems of tilapia. The group supplemented with Chaetoceros sp. powder exhibited a significantly higher respiratory burst activity compared to the control group in both spleen and head kidney (p < 0.05) (Figure 3B,D). Moreover, the experimental outcomes also revealed that the addition of Chaetoceros sp. positively influenced the respiratory burst activities of red hybrid tilapia in a dose-dependent manner, although no significant variations were observed between fish supplemented with 2% or 5% of Chaetoceros sp.

3.3. Lymphoproliferation Activity

Following this, the proliferative capacities of spleen and head kidney B-lymphocytes stimulated with Chaetoceros sp. were investigated and presented in Figure 4. Secondly, the proliferative responses of T-lymphocytes of red tilapia were also evaluated, as shown in Figure 5. In Phase One, the concentrations of 4, 8, and 10 mg/mL of Chaetoceros sp. extract were used in conjunction with results from the lysozyme assay to further evaluate their effects. Both organs were exposed to the mitogens Escherichia coli O111:B4 lipopolysaccharide (LPS) and Concanavalin A (Con A), individually, to activate the B-lymphocytes and T-lymphocytes, respectively.

Based on the results observed in Phase One, the spleen B-lymphocyte proliferations (Figure 4A) showed significant differences among the concentrations upon activation by LPS (p < 0.05). In particular, the post hoc analysis revealed that the concentration of 8 mg/mL Chaetoceros sp. extract differed significantly from the control and the other dosages (4 mg/mL and 10 mg/mL). Conversely, there were no significant variations observed in the head kidney B-lymphocyte proliferation (Figure 4C). Meanwhile, we can observe in Figure 5C that the head kidney T-lymphocytes demonstrated significant variations in proliferation upon Con A activation at 8 mg/mL of Chaetoceros sp. extract, alongside the 10 mg/mL concentration, which also showed a difference when compared to the control group (p < 0.05). It is noted, however, that there was no significant difference in the T-cell proliferation of the spleen among the concentrations tested (Figure 5A).

On the other hand, the Phase Two trial reveals that spleen B-cells’ responses to LPS stimulation were significantly increased (p < 0.05) in the group supplemented with 5% of Chaetoceros sp. compared to the control group and the 2% Chaetoceros sp. powder group (Figure 4B). The spleen T-cell activity towards Con A activation was significantly enhanced (p < 0.05) in fish fed with Chaetoceros sp. powder, highlighting the immunomodulatory potential of this microalga (Figure 5B). However, no significant difference was observed between the two doses of Chaetoceros sp. (p > 0.05). Moreover, the stimulation index of head kidney T-lymphocytes (Figure 5D) was remarkably enhanced in the group supplemented with 5% of Chaetoceros sp. compared to the control group (p < 0.05). Meanwhile, the B-cells in the head kidney (Figure 4D) displayed a dose-dependent increase, although no significant differences were observed between the administered dosages (p > 0.05).

3.4. Growth Performance

The growth performance parameters of red hybrid tilapia during the feeding trial, including measurements at Week 4 and at the end of the 8-week feeding period, are presented in

Table 3. Growth parameters and survival of red hybrid tilapia upon supplementation of Chaetoceros sp.

Parameters	Experimental Group	4 Weeks Post-Feeding	8 Weeks Post-Feeding
Average weight (g)	Control	28.08 ± 0.58	45.81 ± 1.57
	2% microalga	25.67 ± 0.08	49.26 ± 1.58
	5% microalga	28.04 ± 1.09	47.1 ± 2.74
Weight gain (g)	Control	14.08 ± 0.58	31.81 ± 1.57
	2% microalga	11.67 ± 0.08	35.26 ± 1.58
	5% microalga	14.04 ± 1.09	33.10 ± 2.74
Specific growth rate, SGR *	Control	2.44 ± 0.07 *	2.11 ± 0.06
	2% microalga	2.11 ± 0.01 *	2.24 ± 0.06
	5% microalga	2.4 ± 0.16 *	2.15 ± 0.10
Mean survival (%)	Control	95 ± 2.89	90 ± 4.08
	2% microalga	95 ± 2.89	95 ± 2.89
	5% microalga	95 ± 5.00	95 ± 5.00
Feed conversion ratio, FCR	Control	1.75 ± 0.04 a	1.98 ± 0.11
	2% microalga	2.18 ± 0.03 b	1.80 ± 0.08
	5% microalga	1.9 ± 0.26 ab	1.96 ± 0.15
Total yield (g)	Control	224.67 ± 4.63	426.67 ± 6.57
	2% microalga	205.33 ± 0.67	476.33 ± 24.32
	5% microalga	224.33 ± 8.69	471.00 ± 27.39
Hepatosomatic index, HSI	Control	-	2.35 ± 0.19 a
	2% microalga	-	1.66 ± 0.10 b
	5% microalga	-	2.25 ± 0.14 ab
Survival after bacterial challenge (%)	Control	-	46.67 ± 0.33 a
	2% microalga	-	70.00 ± 0.58 b
	5% microalga	-	53.33 ± 0.33 ab

Note. Data are expressed as mean ± SEM. Red tilapia were challenged with 4.5 × 10⁸ CFU/mL of Streptococcus agalactiae. Superscript ^a,b indicates a significant difference between groups (p < 0.05, n = 3). Asterisk * indicates a significant difference in the parameter. “-” indicates parameters not determined at 4 weeks, as fish were not sacrificed and bacterial challenge was only conducted after the 8-week feeding period.

.

Upon the 4th week of Chaetoceros sp. supplementation to red tilapia, there were parameters which showed significant variations between groups. Notably, there were significant differences in specific growth rate (Welch’s test; p < 0.05) of tilapia; the Games–Howell post hoc test did not show variation between groups. Besides that, the FCR during the 4th week of supplementation demonstrated significant differences between groups (Welch’s test; p < 0.05). Specifically, the Games–Howell post hoc test revealed that the 2% Chaetoceros sp. differed significantly from the control group. No significant variations (Kruskal–Wallis; p > 0.05) were observed in the average weight, weight gain, mean survival and total yield of tilapia enriched with microalgae on the 4th week trial.

Furthermore, the growth parameters of red tilapia fed with Chaetoceros sp. were assessed at the 8th week of supplementation. No significant differences (Kruskal–Wallis and One-way ANOVA; p > 0.05) were shown in the average weight, weight gain, average survival, FCR, SGR, and total yield of tilapia. Significant variation was illustrated in the hepatosomatic index (One-way ANOVA; p < 0.05) in which the 2% Chaetoceros sp.-enriched group differed significantly from the control group, as determined using Tukey’s HSD post hoc test.

The survival of red hybrid tilapia after being challenged with S. agalactiae is presented in Figure 6. The group fed with 2% Chaetoceros sp. showed a significantly higher survival rate (70.0 ± 0.58%) compared to the control group (46.7 ± 0.33%) (p < 0.05). The 5% Chaetoceros sp. group had a survival rate of 53.3 ± 0.33%, which was not significantly different from the control group. The Kaplan–Meier survival curve illustrates the progression of mortality over the 14-day challenge period.

4. Discussion

The purpose of this study was to improve understanding of the potential role of Chaetoceros sp. in modulating innate and adaptive immune responses in red hybrid tilapia. Phase One was designed as a preliminary in vitro screening process to evaluate immunomodulatory activity using a Chaetoceros sp. methanolic extract prior to dietary supplementation. This approach supports concentration selection for subsequent feeding trials and reduces the need for extensive animal sampling at the screening stage [69]. Compared with whole microalgal cells, extracts provide more direct exposure of immune cells to soluble bioactive constituents, including antioxidant and anti-inflammatory metabolites. Extracts from marine algae have also been reported to exert immunostimulatory and antiviral activities in mammalian cell systems [70,71,72]. In addition, intact microalgal cells may limit assay responsiveness because rigid cell walls can restrict the release and availability of intracellular compounds [73,74]. Consequently, extract-based screening offers a practical strategy for identifying concentrations with measurable immunological effects under controlled conditions.

It should be acknowledged, however, that leukocytes used in Phase One were cryopreserved prior to functional assays. Although 10% DMSO-based cryopreservation is widely applied in immunological studies [49,58] and post-thaw viability was standardised before experimentation, freeze–thaw cycles may influence certain cellular functions and potentially affect absolute response magnitudes. Therefore, the Phase One findings should be interpreted primarily as comparative screening data for concentration selection rather than as definitive indicators of in vivo immune status. Future investigations would benefit from the use of freshly isolated cells and the inclusion of expanded innate immune assays to further validate functional outcomes.

Functional assays are commonly used to examine cellular responses following exposure to algal-derived extracts across multiple cell types, including fish liver cells [75], mammalian cells [76], gill cells [61], and human cancer cells [62,63]. In Phase One, 8 mg/mL Chaetoceros sp. extract emerged as a promising concentration with consistent immunological modulation. At this concentration, lysozyme activity in blood serum and spleen leucocytes increased significantly, and lymphoproliferative activity was enhanced in head kidney T-lymphocytes and spleen B-lymphocytes. Although higher concentrations increased selected parameters (for example, spleen lysozyme activity at 10 mg/mL), 8 mg/mL was prioritised to achieve balanced immune modulation while avoiding unnecessary escalation in dosage.

Phase Two evaluated immune responses following continuous dietary supplementation with Chaetoceros sp. powder over an 8-week feeding period, with interim assessment at Week 4 and final assessment at Week 8. The findings indicate that dietary Chaetoceros sp. modulated immunity, with significant differences relative to the control group observed in lysozyme and respiratory burst activities in spleen and head kidney, and in splenic T-cell proliferative responses.

Fish innate immunity is often assessed using lysozyme activity, as lysozyme plays a major antimicrobial role against both Gram-positive and Gram-negative bacteria [64]. Accordingly, lysozyme activity is frequently used as a proxy for overall immunocompetence and to evaluate functional feed additives, including microalgae [65,66]. In Phase One, lysozyme activity in serum (Figure 2A) and spleen leucocytes (Figure 2C) was significantly enhanced at 8 mg/mL extract, consistent with reports describing immune potential of algal methanolic extracts [77,78]. Increased lysozyme activity is commonly associated with improved resistance to infectious agents [79].

However, head kidney-derived leucocytes did not show significant variation in lysozyme activity across tested extract concentrations (Figure 2E). In addition, lower extract concentrations (0.8–2 mg/mL) were associated with reduced serum lysozyme activity relative to the control. Lysozyme responses in vitro can be influenced by multiple experimental and biological factors [80,81], and organ-specific immune compartmentalisation may also contribute to differential sensitivity between spleen and head kidney leucocytes. Similar patterns have been reported, including reduced lysozyme responses in Senegalese sole following exposure to polysaccharide-enriched microalgal extracts [82]. High antioxidant content may also reduce measured lysozyme activity through interactions that affect enzymatic function [83]. Extracts rich in reactive compounds have been reported to inhibit lysozyme activity under in vitro conditions [84,85]. Collectively, these observations suggest that the extract may exert mixed effects depending on concentration and cellular context, and that antioxidant-associated interactions may partly explain reduced or unchanged lysozyme activity in certain assays.

In Phase Two, dietary supplementation supported the hypothesis that Chaetoceros sp. can elevate lysozyme activity in red hybrid tilapia (Figure 2), consistent with reports on other diatoms influencing fish immune responses [15,86]. Microalgae contain multiple bioactive components implicated in immune modulation, including sulphated polysaccharides [87,88], β-1,3-glucans [89], selected fatty acids [90], and long-chain polyunsaturated fatty acids such as DHA and EPA [91]. Such constituents have been reported in several diatom genera including Chaetoceros, Navicula, Amphora, Conticribra, and Thalassiosira [37,92,93,94,95,96]. Although specific profiling was not performed here, previous work has shown that diets enriched in DHA/EPA-containing microalgae can enhance lysozyme activity in tilapia [97]. Related findings have been reported elsewhere [98,99,100], although not universally [101,102], indicating that efficacy can be species-, dose-, and context-dependent. The importance of optimal dosing has been emphasised for immunomodulators such as β-glucans [103,104], and dietary fatty acid balance has also been linked to immune outcomes in fish [102]. While β-1,3-glucans are frequently highlighted as potent innate immunostimulants in fish, particularly for enhancing macrophage activation and phagocytic responses, the present study utilised crude extract and whole microalgal biomass rather than purified fractions. Therefore, attributing the observed survival enhancement to a single compound would be speculative. It is more likely that the immunological effects result from synergistic interactions among multiple bioactive constituents, including sulphated polysaccharides, β-glucans, polyunsaturated fatty acids, and carotenoids. Future studies involving targeted compound profiling, fractionation, and metabolomic characterisation would be necessary to identify the primary drivers underlying the enhanced disease resistance observed.

When comparing phases, higher apparent lysozyme ranges were observed during Phase 1 screening than during Phase 2 feeding assessments. One interpretation is that extract exposure in vitro provides immediate access to soluble constituents, whereas whole-cell supplementation in vivo may yield different bioavailability, digestion-mediated release, and systemic regulation. Whole microalgal cells also provide a broader suite of components that may act synergistically over time [105,106]. Consistent with this possibility, whole-cell diatom treatments have been reported to elicit stronger lysozyme responses than corresponding extracts in some species [82]. These considerations suggest that extract screening and dietary delivery may produce different response magnitudes and kinetics despite indicating a broadly similar direction of immune modulation.

A non-linear dose response was also observed in Phase Two, with the 2% group showing comparable or higher activity than the 5% group in some parameters, despite the absence of statistical significance. Similar non-linear patterns have been documented for microalgal or derived immunostimulants across fish species and inclusion levels [31,89,107,108]. Such outcomes may reflect saturation thresholds, regulatory feedback mechanisms that maintain immune homeostasis, or mild inhibitory effects at higher inclusion levels [109,110]. Together, these findings support the interpretation that immunomodulatory effects may be optimised within a moderate inclusion range, rather than increasing proportionally with dose.

Respiratory burst activity is another indicator of innate defence, reflecting reactive oxygen species (ROS) generation by phagocytic cells during antimicrobial responses [111,112,113,114]. In Phase One, respiratory burst activity did not differ significantly across extract concentrations (Figure 3), consistent with reports where microalgae-derived products did not enhance this parameter [89,115]. This may relate to antioxidant constituents such as fucoxanthin, which can scavenge ROS and thereby reduce detectable ROS generation under stimulation [116,117]. Fucoxanthin-mediated inhibition of ROS has been documented in multiple experimental systems [118,119,120,121,122,123], and high antioxidant capacity has also been reported for Chaetoceros extracts [42,124,125]. More broadly, antioxidant activity can influence inflammatory signalling because ROS function as secondary messengers in inflammatory gene regulation [126,127,128,129]. The anti-inflammatory potential of methanolic extracts has been reported for diverse natural sources, including algae and plants [130,131,132,133,134,135,136], and several algae-derived compounds (including fucoxanthin, carotenoids, EPA, DHA, and phenolics) have been linked to anti-inflammatory effects [137,138,139,140,141,142,143,144,145,146,147,148]. Therefore, the absence of significant respiratory burst enhancement in Phase One may be compatible with antioxidant-driven modulation rather than a lack of immunological activity.

In contrast, Phase Two dietary supplementation produced significantly higher respiratory burst activity than the control group, suggesting that sustained intake and physiological processing may promote immune readiness in vivo. Longer exposure can support assimilation and accumulation of bioactives, potentially revealing effects that are not captured in short-term in vitro assays [149,150]. Enhanced respiratory burst activity may strengthen early antimicrobial defence through increased ROS-mediated bacterial killing mechanisms [151,152], and ROS signalling can also interact with adaptive immune activation pathways, including lymphocyte responses [153,154]. These outcomes support the potential of dietary Chaetoceros sp. as a functional ingredient for improving immune responsiveness in red hybrid tilapia.

Lymphocyte proliferation is frequently used to evaluate adaptive immune responsiveness. T-lymphocytes typically respond to mitogens such as PHA and Con A [155,156,157], whereas B-lymphocytes are commonly stimulated by LPS [158,159]. In Phase One, significant differences were detected in splenic B-cell SI at 8 mg/mL extract and in head kidney T-cell SI at 8–10 mg/mL, indicating concentration-dependent modulation. Comparable patterns have been reported in other immunomodulation studies, including dose-dependent effects observed only at higher exposure ranges [160,161], and enhanced leucocyte proliferation in tilapia following microalgal supplementation [49]. Additional evidence suggests that low concentrations may trigger regulatory signals that suppress proliferation, while higher concentrations may activate alternative pathways or accessory cell functions that enhance proliferation [162], which may help explain reduced SI at lower extract concentrations.

In Phase Two, splenic responses to LPS and Con A differed by inclusion level, with enhanced B-cell proliferation at 5% and enhanced T-cell proliferation at both 2% and 5%. Similar enhancement of Con A- and LPS-stimulated proliferative responses has been reported with other natural immunomodulators [163], although inconsistent effects across studies also highlight species- and context-specific variability [164,165]. Head kidney lymphocyte SI showed a dose-dependent trend without statistical significance, consistent with reports in tilapia and other fish where proliferative responses increased modestly with supplementation but did not always reach significance [59,166].

Growth performance data indicate that fish fed 2% and 5% Chaetoceros supplementation showed comparable or slightly lower performance than the control diet at Week 4, but improved or equivalent outcomes by Week 8. Such transient early responses may be linked to palatability changes and an adaptation period to novel dietary components, particularly when experimental fish were previously acclimatised on diets without microalgae [167]. In addition, early immune activation may impose a metabolic cost, as the stimulation of innate and adaptive immune pathways requires energy for leukocyte proliferation, synthesis of antimicrobial proteins, and oxidative defence mechanisms. During the initial supplementation period, this immune-related energy allocation may temporarily divert resources away from somatic growth. Longer feeding durations may permit physiological adaptation, stabilisation of immune responses, and improved utilisation of beneficial constituents, as reported for other novel functional feed components [168]. Although several growth parameters did not reach statistical significance by Week 8, the overall pattern suggests that longer trials may be required to fully capture growth-related effects.

Additional endpoints at Week 8, including hepatosomatic index and post-challenge survival following Streptococcus agalactiae infection, provide further insight into physiological and immune status. A significantly lower hepatosomatic index in the 2% group may suggest improved nutrient utilisation and reduced hepatic lipid deposition. HSI is widely used as a feeding-related indicator reflecting changes in liver energy reserves and mobilisation [169,170,171]. In parallel, increased respiratory burst activity and lysozyme activity in immune tissues support enhanced innate defences that may contribute to improved disease resistance. Similar associations between elevated innate immune parameters and increased survival after bacterial challenge have been reported in fish models [172]. Overall, the combined innate and adaptive responses observed here support the potential of Chaetoceros supplementation to strengthen host defences and improve resilience to bacterial infection. From a commercial standpoint, the optimal inclusion level depends on the production objective and the lowest effective dose that yields a consistent benefit without compromising growth performance. In this study, 2% Chaetoceros sp. improved post-challenge survival and enhanced key innate immune parameters, supporting its use as a practical baseline inclusion level. Higher inclusion (5%) increased lymphoproliferative responses, suggesting enhanced adaptive immune stimulation, and may be more suitable as a short-term strategic inclusion during high-risk periods, pending farm-level cost–benefit validation.

5. Conclusions

In conclusion, this study demonstrates that dietary supplementation with Chaetoceros sp. can effectively modulate the immune responses of red hybrid tilapia. The 2% inclusion level was found to be optimal, leading to significant improvements in lysozyme activity, respiratory burst, and survival following a bacterial challenge with S. agalactiae. These findings highlight the potential of Chaetoceros sp. as a natural and effective immunomodulatory feed additive in aquaculture, offering a promising alternative to antibiotics for disease prevention and health management.