All experimental procedures involving Anguilla bicolor bicolor were conducted in accordance with the institutional guidelines for the care and use of aquatic animals in research and were reviewed and approved by the Ethical Committee of the National Research and Innovation Agency (BRIN), Indonesia, under approval number 253/KE.02/SK/11/2024. The approved protocol covered all stages of the study, including the collection, transportation, acclimatization, stocking, rearing, feeding, handling, sampling, and euthanasia of glass eels used in the experiment.

Particular attention was given to minimizing animal stress and unnecessary suffering throughout the study. Glass eels collected from the Cimandiri River estuary were transported in oxygenated double-layer plastic bags placed in insulated containers with ice gel packs to maintain suitable conditions during transit. On arrival at the experimental facility, the fish were acclimatized for 7 days before the feeding trial to allow recovery from transport-related stress. During the 60-day rearing period, fish were maintained under controlled environmental conditions with continuous aeration and water quality kept within acceptable ranges for eel culture. Handling during routine management and sampling was performed carefully and only when necessary to reduce physical disturbance and stress.

At the end of the experiment, fish sampling for growth assessment, histological examination, and molecular detection of AngHV-1 was carried out in accordance with the approved ethical protocol. The number of fish sampled was kept to the minimum required to achieve the scientific objectives while ensuring reliable data collection. Thus, the study complied with recognized animal welfare principles for the humane use of aquatic organisms in experimental research.

The experiment was conducted over a 5-month period from December 2024 to April 2025 at the Brackishwater Aquaculture Development Center (BADC), Jepara Subdistrict, Jepara Regency, Central Java Province, Indonesia (59418).

The inoculum of P. cruentum was obtained from pure stock cultures maintained at BADC, Jepara, during the intermediate (exponential) growth phase in 20 L glass carboys to ensure culture purity and sufficient cell density. Cultures were kept in f/2 medium [12] at 30 g/L (approximately 30 ppt) salinity and 24°C ± 1°C, under a 12 h light: 12 h dark photoperiod with continuous aeration.

Glass eels of A. bicolor bicolor were collected from local fishers operating in the Cimandiri River estuary, Pelabuhan Ratu Subdistrict, Sukabumi Regency, West Java Province, Indonesia. For transportation, the glass eels were packed in double-layer plastic bags containing 3 L of water with a biomass density of approximately 200 g per bag. The bags were oxygenated and placed in insulated foam boxes containing four ice gel packs (500 g; 20 × 15 × 3 cm). No water exchange was conducted during transport. Upon arrival at the experimental facility, the glass eels were acclimatized for 7 days in 0.5 m³ fiberglass tanks to recover from transport stress and were fed Artemia ad libitum.

After acclimation, glass eels were randomly placed into 70 L plastic containers (53.5 × 37.5 × 32.0 cm³) with a water volume of 30 L. The containers were situated in a semi-indoor facility (200 m²) under a 12 h light and 12 h dark photoperiod. Each container had a single aeration point connected to a root blower system.

Water used for the culture system was sourced from a borewell and allowed to settle for 3 days in a 10 m³ aerated concrete tank. The water was then supplied to the experimental tanks using a 30 W submersible pump through dual cartridge filtration units (Kinnoyama, Nanotech @ SG Clear). The initial mean body weight and total length of the glass eels were 0.12 ± 0.01 g and 4.96 ± 0.09 cm, respectively. Fish were stocked at a density of 90 individuals per tank and reared for 60 days under the respective feeding treatments.

Artemia cysts (Supreme Plus, Golden West®, Great Salt Lake, USA) were used as live feed. Cysts were hatched following standard procedures commonly used for commercial Artemia production in marine fish larviculture. Briefly, cysts were incubated in conical hatching tanks containing artificial seawater at 30 g/L salinity and 28°C ± 1°C with continuous aeration and illumination for 24 h to obtain instar-I nauplii [13, 14].

For enrichment, newly hatched nauplii were stocked at approximately 300–500 individuals/L in 5 L cylindrical-conical vessels containing seawater at 30 g/L salinity and 28°C ± 1°C. Strong aeration was provided to maintain suspension of both Artemia and microalgal cells. The experimental design consisted of one control treatment (unenriched Artemia) and six enrichment treatments with different combinations of P. cruentum cell density and enrichment duration: control (no enrichment), A (3 × 10⁵ cells/mL for 6 h), B (3 × 10⁵ cells/mL for 12 h), C (6 × 10⁵ cells/mL for 6 h), D (6 × 10⁵ cells/mL for 12 h), E (9 × 10⁵ cells/mL for 6 h), and F (9 × 10⁵ cells/mL for 12 h). Each treatment was replicated three times (n = 3 tanks). Throughout the experimental period, glass eels were fed exclusively with Artemia corresponding to the assigned enrichment treatment.

At the start of the experiment, 50 fish were randomly sampled to determine initial body weight and total length. At the end of the 60-day trial, all fish from each tank were individually measured to assess final body weight and total length. The growth parameters evaluated included survival rate (SR) [16], average weight gain (AWG) [17], and specific growth rate (SGR).

SR (%) was calculated as: SR = 100 × (Nf/Ni), where Nf represents the final number of fish and Ni represents the initial number of fish.

AWG (g) was calculated as: AWG = Wf − Wi, where Wf and Wi represent the final and initial mean body weights, respectively.

SGR (%/day) was calculated as: SGR = 100 × (ln Wf − ln Wi)/t, where t represents the rearing period (days).

The proximate composition of Artemia (moisture, crude protein, crude lipid, and ash) after enrichment with P. cruentum was determined using standard analytical methods [18–20]. For each treatment, three independent Artemia samples of approximately 30 g wet weight were collected at the end of the enrichment period. The samples were rinsed with distilled water, blotted dry, frozen at −20°C, freeze-dried, and homogenized using a mortar and pestle before analysis [21].

Moisture content was determined according to SNI 01-2891-1992. Crude protein was analyzed using the 18-8-31/MU titrimetric method, whereas crude lipid content was determined using method 18-8-5/MU (Weibull gravimetric method). Ash content was measured following SNI 01-2891-1992 section 6.1. Nitrogen-free extract (NFE, %) was calculated as: NFE = 100 − (moisture + protein + lipid + ash) [22]. Gross energy (kcal/100 g) was estimated using physiological fuel values of 4.0 kcal/g for protein and NFE and 9.0 kcal/g for lipid according to Atwater conversion factors [23, 24].

At the end of the experiment, one glass eel from each replicate tank was sampled for histological examination. Tissue samples were fixed in Bouin’s solution for 48 h and then dehydrated through graded ethanol concentrations (70%, 80%, 90%, 95%, and 100%), followed by immersion in an ethanol–xylol solution (1:1). The tissues were embedded in paraffin blocks and sectioned transversely at the abdominal region (from the posterior part of the stomach to the anus) using a microtome. Histological sections were stained with hematoxylin–eosin.

Microscopic observations were performed using an Olympus BX53 microscope (Japan) at 10× magnification. Villus length and surface area were calculated with the modified formula: villus surface area (µm²) = [(b + c)/2] × a, where a represents villus height or length, b represents apical villus width, and c represents basal villus width.

Detection of AngHV-1 infection in the A. bicolor bicolor glass eel population was carried out twice: once before the experiment began and again at the end of the rearing period. Viral genome detection was performed qualitatively (semi-quantitatively) using conventional polymerase chain reaction based on the method described previously [25], with minor modifications.

For each treatment and sampling time, gill and kidney tissues from five glass eels (one fish from each replicate tank) were pooled to create a single DNA sample. Genomic DNA was extracted using a commercial tissue DNA extraction kit (Geneaid™ DNA Isolation Kit Ver. 06.22.16) following the manufacturer’s instructions. The quality and concentration of the extracted DNA were measured with a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, WA, USA).

Detection of AngHV-1 was carried out using the primer pair HVAPOLVPSD and HVAPOLOOSN (Table 1), which amplify a 394-bp fragment of the viral genome [25]. PCR amplification was performed under the following conditions: initial denaturation at 94°C for 5 min (1 cycle), followed by 40 cycles of denaturation at 94°C for 30 s, primer annealing at 65°C for 45 s, and extension at 72°C for 60 s, with a final extension step at 72°C for 7 min. PCR products were stored at 4°C until analysis and subsequently visualized using 2% agarose gel electrophoresis to confirm the presence of viral DNA bands.

All results are presented as mean ± standard error (SE), with the tank considered as the experimental unit. Before conducting statistical analysis, the data were tested for normality using the Shapiro–Wilk test and for homogeneity of variance with Levene’s test. When these assumptions were met, a one-way analysis of variance was used to assess the effect of treatments. If significant differences were found (p < 0.05), Duncan’s multiple range test was applied as a post hoc test to compare treatment means. Statistical analyses were conducted using SPSS software version 31 (IBM Corp., NY, USA).

The proximate composition and energy content of enriched Artemia are shown in Table 2. Moisture content, expressed on a wet weight basis, ranged from 91.83% to 94.21%, while crude protein, crude lipid, ash, NFE, and gross energy were expressed on a dry matter basis. Compared to the control, enrichment consistently enhanced the nutritional value of Artemia. Crude protein increased from 47.72% in the control to 48.28%–61.48% in the enriched treatments, with the highest level observed in treatment A. Even greater increases were seen in crude lipid, which rose from <0.30% in the control to 4.72%–9.98% in the enriched groups, reaching a maximum in treatment F. This rise in lipid content contributed to higher fat-based energy, which increased from a negligible amount in the control to 3.29–5.67 kcal/100 g in the enriched treatments.

Total gross energy increased across all enriched groups, with the highest values seen in treatments C (26.74 kcal/100 g) and E (26.71 kcal/100 g), compared to 18.90 kcal/100 g in the control. Conversely, ash and NFE showed variable patterns among treatments. These findings suggest that enrichment with P. cruentum enhanced not only the macronutrient profile of Artemia but also its overall energy value.

The significant increase in crude protein and crude lipid highlights P. cruentum’s effectiveness as a bioencapsulation agent. Artemia’s active filtration behavior helps it absorb biomolecules from P. cruentum, including proteins, unsaturated fatty acids, and lipophilic pigments [26]. The biochemical makeup of P. cruentum includes protein (30%–43%), lipid (9%–14%), and EPA, which makes up about 9.7% of total fatty acids [27]. Additionally, compounds like sulfated polysaccharides and phycoerythrin further boost the nutritional and biological value of live feed [28, 29]. Therefore, enrichment with P. cruentum enhances both the quantity and quality of Artemia.

Among the tested treatments, C offered the most balanced nutrient profile, with crude protein at 52.59%, crude lipid at 4.72%, and total energy of 26.74 kcal/100 g. The 6 h enrichment period likely coincided with the peak ingestion phase of Artemia on microalgal cells [26], making it an efficient enrichment window [30]. The increased energy from lipid and pigment fractions, along with the antioxidant and bioactive properties of P. cruentum, may also enhance the immunoprotective value of enriched Artemia [27, 31]. Overall, these findings suggest that enrichment with P. cruentum improves Artemia’s nutritional profile and enhances its suitability as a strategic live feed for the early rearing phase of A. bicolor bicolor.

The survival of A. bicolor bicolor over the 60-day rearing period varied significantly among treatments (Figure 1). The control group had the lowest survival rate at 15.56% ± 5.9%, while treatment C achieved the highest survival at 60.74% ± 3.9%, which was significantly higher than all other groups (p < 0.05). Treatments A (28.89% ± 9.2%), B (20.00% ± 9.5%), D (27.41% ± 9.3%), and F (26.67% ± 6.9%) did not differ significantly from the control (p > 0.05). Treatment E (32.78% ± 2.4%) showed significantly higher survival than the control but remained lower than treatment C. These findings demonstrate that enriching Artemia with P. cruentum at 6 × 10⁵ cells/mL for 6 h was the most effective condition for improving survival during rearing.

The improved survival rate is likely due to the increased supply of nutrients and bioactive compounds transferred from P. cruentum to Artemia. Higher lipid and protein levels provide vital energy and substrates for larval development [32, 33]. Lipids act as a key energy reserve during metamorphosis, while proteins support tissue repair and growth. Additionally, PUFA, especially DHA, along with phycoerythrin from P. cruentum, may enhance energy metabolism, enzymatic activity, membrane fluidity, and osmoregulation [33, 34]. Functional compounds such as sulfated polysaccharides and EPA may also boost larval immunity and thus improve survival [28, 35].

Final body weight after 60 days also varied significantly among treatments (Figure 2). The control group had the lowest final body weight, at 0.181 ± 0.02 g, while treatment C showed the highest value, at 0.250 ± 0.01 g, which was significantly higher than all other treatments (p < 0.05). Treatments A (0.197 ± 0.03 g), B (0.199 ± 0.03 g), D (0.203 ± 0.02 g), E (0.191 ± 0.02 g), and F (0.186 ± 0.02 g) did not differ significantly from the control (p > 0.05). These results suggest that treatment C was also the most effective in increasing final body weight.

A similar pattern was observed for SGR (Figure 3). Treatment C yielded the highest SGR, at 1.11 ± 0.1%/day, which was significantly greater than the control (0.65 ± 0.1%/day) and all other treatments (p < 0.05). In contrast, treatments A (0.69 ± 0.2%/day), B (0.59 ± 0.6%/day), D (0.82 ± 0.1%/day), E (0.84 ± 0.1%/day), and F (0.80 ± 0.2%/day) did not differ significantly from the control (p > 0.05). Therefore, enriching Artemia with P. cruentum at 6 × 10⁵ cells/mL for 6 h produced the greatest improvement in both final body weight and SGR of A. bicolor bicolor.

These growth responses align with the nutrient profile shown in Table 2. Treatment C offered a balanced mix of crude protein, crude lipid, and total energy, ensuring an adequate supply of amino acids and energy for larval growth. Protein serves as the main substrate for tissue synthesis and enzyme production, while lipid, especially PUFA, functions as a key energy reserve during metamorphosis and developmental transitions [32, 33]. Since zooplankton naturally provide a major lipid source for larvae, enriching Artemia with P. cruentum may better mimic natural feeding conditions by increasing the availability of long-chain fatty acids essential for larval nutrition [33, 34].

The physiological value of P. cruentum is further supported by its high content of PUFA, including EPA and DHA precursors, as well as phycoerythrin and sulfated polysaccharides. These compounds may boost membrane fluidity, metabolic efficiency, osmoregulation, and immune responses [28, 34, 35]. Transferring these compounds into Artemia enhances the functional quality of the live feed by providing not just energy but also biologically active substances that aid larval physiology and resilience [36–38]. The simultaneous increase in survival and growth suggests that nutritional improvements benefited both parameters at the same time. Since these gains were not linked to changes in intestinal villus morphology, the improved performance in treatment C seems to be mainly due to better nutrient availability and bioactive quality rather than structural modifications of the digestive tract.

Histological examination of the intestine at the end of the rearing period mostly showed uniform villus structure across treatments (Figure 4). No significant differences were seen between the control and enriched groups. This was supported by morphometric analysis, where villus length ranged from 95.00 to 147.06 µm and villus surface area ranged from 3,717.25 to 6,527.76 µm², with no significant differences among treatments (p > 0.05) (Figure 5). These results suggest that enrichment with P. cruentum, regardless of cell density or duration, did not change intestinal histology, and the basic digestive structure remained similar across treatments.

Intestinal villi are finger-like projections that enhance the surface area for absorption, thereby aiding in the uptake of nutrients and water. Villus height and surface area are frequently used as markers of nutrient absorption ability [39]. Increased villus height, fold number, and surface area generally correlate with better nutrient absorption and utilization because a larger epithelial surface is available for diffusion and active transport [40].

In the present study, although enrichment with P. cruentum significantly enhanced the growth performance of A. bicolor bicolor, no corresponding changes were observed in villus morphometry. Villus length and surface area remained relatively unchanged across treatments, indicating that dietary enrichment did not cause histological adaptation in the intestine. Therefore, the increased growth seen in treatment C cannot be attributed to an expanded intestinal structural capacity.

These results indicate that P. cruentum mainly contributed by increasing metabolic energy availability rather than altering intestinal structure. Red microalgae are rich in polysaccharides, proteins, and functional lipids such as PUFA, which can boost energy density and metabolic efficiency in larvae [22, 34]. Therefore, the extra energy supplied through enriched Artemia was more likely used for somatic growth and metabolic activities without changing villus morphology. In summary, P. cruentum enrichment seems to support the growth of A. bicolor bicolor by improving nutrient and bioactive compound availability while keeping the intestinal structure stable.

Water quality remained fairly constant across treatments during the rearing of A. bicolor bicolor (Table 3). DO ranged from 4.60 to 6.50 mg/L, temperature from 26.80°C to 28.00°C, pH from 7.26 to 8.55, and total ammonia nitrogen (TAN) from 0.001 to 0.448 mg/L. These values stay within acceptable ranges for the growth and development of A. bicolor bicolor and suggest that no significant environmental fluctuations took place during the culture period. This stability also indicates that using Artemia enriched with P. cruentum did not negatively impact water quality.

High-quality and digestible live feed like Artemia is known to reduce uneaten feed residues and nitrogenous waste, thereby decreasing the risk of water pollution [41]. In this study, enrichment with P. cruentum not only enhanced the nutritional profile of Artemia, including water-soluble proteins, PUFA, and antioxidants, but also seemed to support environmental stability. Red microalgae are recognized for their ability to absorb nitrogen, which may indirectly help keep TAN within safe limits [42]. Additionally, several compounds in P. cruentum have antioxidant, anti-inflammatory, and immunomodulatory properties that could reduce stress and assist in maintaining physiological homeostasis in cultured fish [43, 44].

This observation aligns with previous reports suggesting that supplementing aquafeeds with microalgal products can enhance not only nutritional quality but also fish health and water quality stability [45, 46]. Therefore, maintaining suitable water quality in this study probably reflects a positive interaction between the improved nutritional performance of enriched Artemia and the ecological contribution of P. cruentum in the rearing system.

Although water quality stayed within acceptable limits, microbiological conditions also significantly influenced the culture environment. Total plate count (TPC) analysis revealed that bacterial counts were higher in all enriched treatments compared to the control (0.84 × 10⁵ CFU/mL), with the highest count found in treatment D (2.00 × 10⁵ CFU/mL). The other enriched groups ranged from 1.00 × 10⁵ to 1.80 × 10⁵ CFU/mL (Figure 6). Despite this increase, all counts remained within levels typically regarded as safe for cultured fish.

The higher bacterial counts in treatments receiving P. cruentum-enriched Artemia may be attributed to two factors. First, enriched Artemia might have carried additional biomolecules, such as water-soluble proteins, sulfated polysaccharides, and lipophilic pigments, which can serve as substrates for heterotrophic bacteria [36, 38]. Second, red microalgae like P. cruentum are known to produce EPS [35], which may act as carbon sources for microbial communities while also providing immunostimulatory effects in fish.

Moderate bacterial abundance may not necessarily be harmful. Non-pathogenic bacterial communities at low to moderate levels can prevent colonization by pathogenic bacteria through competition for nutrients and space, while also producing antagonistic bioactive metabolites [47]. Therefore, the increase in TPC observed in enriched treatments should not be seen only as a negative result but may instead reflect a more active and potentially beneficial microbial environment.

Importantly, the increased bacterial load did not harm growth performance or survival. This indicates that the beneficial nutritional and bioactive properties of P. cruentum outweighed any potential risks linked to higher bacterial levels. Therefore, enrichment with P. cruentum not only enhanced the nutritional quality of Artemia but may also have affected microbial interactions in the culture system in a way that is compatible with glass eel rearing.

Molecular screening conducted before the experiment confirmed that the A. bicolor bicolor population used in this study was already infected with AngHV-1 (Figure 6). This finding aligns with previous reports showing that wild-caught glass eels commonly harbor viral infections, which pose a major challenge for aquaculture [48, 49]. Semi-quantitative assessment based on DNA band intensity indicated that infection levels were already high at the start of rearing.

After 60 days, polymerase chain reaction analysis showed that AngHV-1 genomic DNA was still detectable in all experimental groups, although band intensity varied among treatments (Figure 7). The control and treatments A, B, D, E, and F exhibited relatively thick bands, indicating severe infection. Treatment B had a moderate band intensity, while treatment C showed only a faint band, consistent with mild infection. These findings suggest that dietary supplementation through Artemia enriched with P. cruentum may lessen the severity of AngHV-1 infection, although it does not completely eliminate the virus.

This pattern can be understood within the framework of herpesvirus biology. As part of the Herpesviridae family, AngHV-1 establishes lifelong latency, with the viral genome remaining in host cell nuclei as episomes and reactivating under stress or immunosuppression [50]. Therefore, although viral DNA was detectable in all treatments, the fainter bands in treatments C and B indicate lower viral activity and suggest the virus may be in a more latent state. This suggests that improved physiological and immunological health in the enriched groups may have suppressed viral replication.

These results also align with previous reports indicating that AngHV-1 is widespread among various Anguilla species, including A. anguilla, A. japonica, A. rostrata, A. bicolor bicolor, and A. marmorata [48, 49, 51–54]. Since artificial propagation of glass eels is not yet available [55, 56], eel aquaculture still relies on wild-caught seed that may already carry infections before entering culture systems. Given these circumstances, completely preventing infection is unrealistic, and management should focus on minimizing viral impact rather than stopping exposure.

Since herpesviruses are obligate intracellular pathogens, direct chemotherapeutic control is highly limited [48]. Therefore, managing AngHV-1 must depend on non-drug approaches. These can include functional feeds and immunostimulants, vaccination, and environmental adjustments such as temperature control to inhibit viral replication [52]. Elevated temperatures have also been reported to boost antiviral responses by increasing the expression of immune-related genes, including interferon-γ, interleukin-1β, and Mx1 [20]. While these methods may not eradicate the virus, they can decrease viral replication and reduce the risk of transmission.

In the present study, the relationship among survival (Figure 1), growth performance (Figures 2 and 3), and AngHV-1 band intensity (Figures 7 and 8) indicates that enrichment with P. cruentum supports the physiological condition of A. bicolor bicolor in a way that promotes improved performance while reducing apparent viral severity. Collectively, these results suggest that nutritional management through P. cruentum bioencapsulation plays a key role in mitigating infection severity and maintaining the performance of A. bicolor bicolor glass eels during culture, even though AngHV-1 remains detectable in all groups.

Several limitations should be considered when interpreting the present findings. First, AngHV-1 load was assessed using conventional polymerase chain reaction and semi-quantitative visual evaluation of band intensity, without employing an internal control gene or quantitative PCR-based quantification. Therefore, the differences in AngHV-1 levels among treatments should be regarded as approximate and require confirmation using more sensitive, quantitative molecular methods. Second, intestinal histological assessment was performed on a limited number of fish and villi per treatment, which may have reduced the ability to detect subtle morphometric differences and could explain the lack of significant variation in villus structure despite the notable improvements in survival and growth.

Third, only total heterotrophic bacterial counts were measured, without distinguishing beneficial bacteria from potentially pathogenic taxa. Additionally, water quality was evaluated at specific sampling points rather than continuously, which limited the temporal resolution of microbial and environmental fluctuations during the culture period. Finally, the experiment was conducted at a pilot semi-indoor scale for 60 days with a single cohort of glass eels from one geographic source. Therefore, caution is necessary when extrapolating these findings to other eel stocks, hatchery systems, or long-term production conditions.

Future research should quantify AngHV-1 dynamics using quantitative PCR and include immune-related endpoints, such as the expression of antiviral and pro-inflammatory genes. It should also incorporate controlled challenge studies to better clarify how P. cruentum-enriched Artemia affects viral replication and host defense. A more comprehensive histological and functional assessment of the digestive tract, including digestive enzyme activity and gut barrier markers, would also help differentiate the relative roles of structural and metabolic factors in improving growth performance.

Additionally, more detailed profiling of crude lipid, fatty acids, and pigments in enriched Artemia, along with characterization of related microbial communities, would offer a more thorough understanding of how P. cruentum alters the nutritional and microbial environment of glass eels. Finally, validation in commercial hatchery settings, including economic analysis and integration with complementary management strategies such as temperature control or vaccination, is necessary to assess the practicality of P. cruentum-enriched Artemia as a non-pharmaceutical approach for managing AngHV-1 in tropical eel aquaculture.