Effects of dried microalgal supplement on molting efficacy, enzyme activity and histological improvement in freshwater prawn (Macrobrachium rosenbergii) larval development: a comprehensive study

Highlights

• •Microalgae supplementation significantly increased larval survival (>90%) in prawn hatchery conditions.
• •Post-larval production was higher in microalgae-fed groups than in the control system.
• •Digestive enzymes (amylase, protease, lipase) were significantly enhanced by microalgal feeding.
• •Hepatopancreas and muscle tissues showed improved structural integrity with reduced degeneration.
• •Microalgae improved larval development synchrony and accelerated molting progression.

Abstract

This research, pioneering in its nature, examined the impact of microalgal supplements on the larval developmental processes and mass production of Freshwater Prawn (Macrobrachium rosenbergii) post-larvae (PL) on a commercial scale. Overcoming the obstacles to prawn PL production in Bangladesh since 2011 was a major focus of our initiative. A total of six cemented tanks, each with a capacity of 3000 liters, were used for the two treatments and a control group in replicates. In Treatment 1 (T1), microalgal (powder) supplementation as nutritional source was administered at a concentration of 4 ppm, consisting of 2 ppm of spirulina powder (SP) and 2 ppm of black algae powder (BAP), four times daily; conversely, Treatment 2 (T2) received the identical dosage six times daily. The control tank (C) did not receive any algal supplementation. PL production, survival rate, digestive enzyme activities, superoxidase dismutase (SOD) activity and histological changes in the hepatopancreas and mussel of the larvae were evaluated to determine the efficacy of the treatments. Approximately, 87,000 freshly hatched larvae were stocked in each tank. Significantly higher (p < 0.05) PL production was found in T2 followed by T1and C. Analogously, a substantially higher survival rate was recorded in T2 (93.59±2.82%) followed by T1 (86.28±3.58%) and C (62.42±0.75%). Amylase enzyme activity was significantly higher (p < 0.05) in T2 followed by T1 and C. Protease and lipase enzyme activity were significantly higher (p < 0.05) in the treatments than the control, however, no significant difference within the treatments was observed. In contrast, SOD activity recorded significantly higher (p < 0.05) in the control than the treatments. Histological evaluations of the hepatopancreas and muscle tissues indicated improved organization and health conditions in PL from treatment tanks, contrasting with irregular distribution and less organized muscles in control PL. The study found that applying 4 ppm microalgae (SP+BAP) 6 times a day can significantly increase healthy PL production in prawn hatcheries.

1. Introduction

Macrobrachium rosenbergii (giant freshwater prawn), locally known as Galda is likely one of the most commercially prudent aquaculture species in the world, mainly, in China, Bangladesh, Thailand, Myanmar, and India. This species contributed $ 2.45 billion to the world economy with a worldwide production of 284,635 MT in 2021 (Pillai & Panda, 2024). The conducive environment and atmosphere of Bangladesh facilitated the expansion of M. rosenbergii farming over an area of 71,770 hectares, resulting in a production of 58,273 t in 2022 (Statistical Yearbook Bangladesh 2023, 2024).

The annual average demand for prawn (M. rosenbergii) post-larvae (PL) in Bangladesh is around 3000 million. Considering the scope of the industry >70 hatcheries were established in the early 20th century to meet the demand for prawn PL in the country (Islam et al., 2018). However, since 2011, mass larval mortality and seize moulting drastically reduced the number of actively PL-producing hatcheries below 80% ( Alam et al., 2019; Hooper et al., 2020; Islam et al., 2018). In the year 2009, a total of 60 prawn hatcheries produced 200 million post-larvae (PL) (Biswas, 2021), which subsequently declined to 49.6 million in the year 2023 (DoF, 2024). Since the catastrophe break out, hatcheries mostly experienced mass larval mortality between III∼IV & VI∼IX stages of larval development and severe seize moulting after stage IX along with anomalous morphologies, poor feeding affinity and a gradual whitening of the body (Alam et al., 2019; Islam et al., 2018; Wahab et al., 2012).

Several national and international organizations and universities including the Shrimp Research Station of Bangladesh Fisheries Research Institute initiated several interventions to overcome the issues (Alam et al., 2019; Biswas, 2021; Islam et al., 2019). Modification to the existing hatchery operation through a rigorous intervention by Islam and his colleagues resolved the mass larval mortality in between the larval development stages and partially solved the PL production hindrance by up to 50% (Islam et al., 2019). To ensure a sustainable solution to the decade-long constraint, the focus has been given to microalgae supplementation during the larval rearing period. Microalgae have been a reliable source of nutrition for larval bivalves, shrimps, and fish both directly and indirectly by the enrichment of rotifers, artemia, and copepods (Ma & Hu, 2024). In hatchery-based seed production, commonly used microalgae such as Chlorella, Nannochloropsis, Isochrysis, Tetraselmis, and Chaetoceros have been reported to improve larval performance by supplying digestible protein, essential fatty acids, carotenoids, vitamins, minerals, and bioactive compounds required for early development, moulting, stress tolerance, and survival (Nagappan et al., 2021; Silveira Júnior et al., 2019; Vijayaram et al., 2024). Moreover, microalgae supplements synthesize several crucial biological elements, viz., amino acids, vitamins, antioxidants, and pigments to enhance metabolic activity and growth in aquatic animal (Chen et al., 2020). On the other hand, Black Algae Powder, a blend of spirulina and beta-carotene applied in the shrimp and fish production industry as a guaranteed product for higher growth and survival rate (Mimbela‐Ipanaque et al., 2025a). Algal and spirulina-based supplementation has also been reported to improve growth performance, feed conversion, antioxidant status, immune response, and disease resistance in several cultured aquatic animals, including Nile tilapia, carp, penaeid shrimp, and freshwater prawn (Amer, 2016; Li et al., 2022; Ringø, 2025) . For instance, dietary spirulina meal significantly improved growth, survival, and feed utilization in Macrobrachium rosenbergii, while spirulina supplementation in fish and shrimp has been described as a promising growth-promoting feed additive (Alagawany et al., 2021; Mimbela‐Ipanaque et al., 2025a). Penaeid shrimp species such as Litopenaeus vannamei and Penaeus monodon, marine fish including species like Oreochromis niloticus (Nile tilapia in marine aquaculture contexts), and bivalves such as Crassostrea gigas (Pacific oyster) and Mytilus edulis have all been widely reported to benefit from microalgal supplementation in seed production systems (Iba et al., 2014; Ronquillo et al., 2012; Silva et al., 2022) . Considering these established benefits of microalgae in penaeid shrimp, marine fish, and bivalve aquaculture, this study aimed to evaluate the effect of algal supplementation on mass-scale post-larval production of Macrobrachium rosenbergii under hatchery conditions. Moulting efficacy, enzyme activity, and histological changes were observed during the study to provide a clear insight into the impacts of microalgae on M. rosenbergii larval development and PL production. To our knowledge, this is the first reported commercial-scale experimental trial on prawn PL production using microalgae.

2. Materials and methods

2.1. Preparation of the experimental tanks

The larvae rearing for the experimental period was carried out in the Prawn Hatchery of Shrimp Research Station. A total of 6 cemented 3000 L capacity Larvae Rearing Tanks (LRT) were used for the experiment, consisting of a control (C) and two treatments (Treatment T1 and Treatment T2) in replicates. Each tank contained a large number of larvae and represented an independent rearing unit. This design was selected because the study was conducted under large-scale operational hatchery conditions, where the availability of 3000 L cemented larval rearing tanks, space, water management facilities, and hatchery resources limited the number of possible replications. Therefore, two replicates per treatment were used to reflect a realistic practical hatchery setup while maintaining separate tank-level treatment units. Water purification and conditioning were performed as previously described by Islam and colleagues (H. M. R. Islam et al., 2019). Black Algae Powder (BAP) (O.S.I, USA) and Spirulina Powder (SP) (O.S.I, USA) were applied only to the treatment tanks at a rate of 4 ppm (2 ppm BAP + 2 ppm SP), 4 times daily, denoted as T1 and 6 times daily, denoted as T2. The dose was selected based on microalgae supplementation practices reported in crustacean larval culture, where repeated low-dose administration has been shown to improve feed availability and digestive efficiency in prawn/shrimp hatchery systems (Elsayed et al., 2026; Jaseera et al., 2021; Silva et al., 2022), with slight modification to suit commercial hatchery conditions. According to the packaging label of that BAP, the nutritional composition (dry matter basis) was: Moisture (Max):8%, Crude Protein (Min):46%, Fat (Min): 12%, Fiber (Max): 3%, Ash (Max): 15%, HCl Insoluble Substance, max: 2%. Beside the nutritional content of SP was: Moisture (Max): 9%, Crude Protein (Min):60%, Fat (Min): 2%, Fiber (Max): 2%, and Ash (Max): 10%. Briefly, algal powders with the given dose per LRT were taken into a 100-mesh micron filter bag and thoroughly mixed by vigorously squeezing in a plastic bucket containing 4 L 12ppt water. The water containing the microalgae was then equally spread throughout the LRT to ensure homogeneous distribution. Continuous aeration was maintained to prevent SP and BAP settling down at the tank bottom. To normalize the effect of continuous addition of SP and BAP, and to enhance larval molting rate, after every three days of rearing, larvae from each LRT were entirely shifted to a previously prepared and cleaned LRT using a 150–200 µm mesh net to pass out the uneaten artemia and any other particulate matter (SP, BAP, feces, etc.) as per the modified hatchery protocol developed based on our previous intervention (Islam et al., 2019). After shifting the larvae, the tank was disinfected with 1% Cl. The tank wall and bottom were cleaned with a soft-bristled brush to remove residual microalgae and fecal matter left after regular siphoning during the rearing periods, then mopped and air-dried before restocking. All the tanks of control and treatment groups were maintained similarly.

2.2. Larval stocking, feeding, and rearing

Freshly hatched larvae of M. rosenbergii were equally stocked (Treatment tanks containing microalgae) in the LRTs containing 1000 L 12 ppt water. To ensure uniform stocking density across all the LRTs, a standardized volume of larvae was systematically transferred utilizing a pre-calibrated modified 10 ml capacity measuring cylinder featuring a 100-micron mesh net at the terminal end designed to facilitate the expulsion of excess water while concurrently retaining the larvae within the chamber. The cylinder was found to carry around 10,000 larvae per ml volume during repeated trials using freshly hatched larvae. Initially, 8 ml of larvae were transferred into each LRT obtained from 24 mother prawns. The remaining larvae were then equally allotted into the LRTs, receiving 0.7 ml larvae per LRT. Subsequently, the number of larvae was critically measured using a volumetric sub-sampling method (Naegel & Gómez-Humarán, 1998) with slight modification. Briefly, day-aged larvae were sampled 4 times with a 250 ml glass beaker from different depths and locations of an LRT in replicates with mild aeration to ensure homogeneous distribution of the larvae. The samples were counted and calculated to estimate the total number of larvae per liter of water (Fig. 2. A). Considering the initial volume of the LRT, the number of larvae per liter of water was multiplied by 1000L to estimate the total number of larvae per LRT at initial stocking (Fig. 2. B). Stocking density was kept within the maximum range suggested in the previous studies (Nhan et al., 2010). As the rearing day progressed, the volume of water within the LRTs was augmented to preserve an optimal environment conducive to the health of the larvae in the LRT. Newly hatched artemia was administered twice daily at a concentration of 3∼5 nauplii/ml (20∼25 g/ton) throughout the days of rearing (DoR) along with formulated egg custard supplement from the 7th DoR as previously reported by several researchers (Biswas, 2021; Correia et al., 2000; FAO, 2002; Lavens et al., 2000). Egg custard was prepared according to the formulation of De Barros and Valenti (2003) where the nutritional composition (dry matter basis) was reported as : Crude Protein :45.07%, Fat: 22.55%, Minerals: 8.83%, and original dry matter: 18.29%. Before each feeding session, fecal matter, residual feed, and exuviae were meticulously siphoned out. Continuous aeration was maintained to ensure an adequate supply of dissolved oxygen.

Fig. 2.

Representative photomicrograph of different stages of prawn larvae during the rearing period.

2.3. Molting and larval development assessment

Molting is the fundamental requirement of larval development in crustaceans. Prawn larvae undergo a total of 11 distinct developmental phases with successive molting to metamorphose into PL within 23 to 27 days (Kanaujia et al., 2005). To evaluate the efficacy of microalgae supplements, larval growth was observed in terms of attaining these different larval developmental stages at a given date, i.e., the 6th, 12th and 18th days of rearing. Briefly, three 500 ml sterile glass beakers were used to collect larval samples from three different points of a larvae rearing tank (LRT) and kept with adequate aeration. Then 100 prawn larvae were observed under a compound microscope (Euromex, Netherland) for the specific attributes to define the stages of the collected sample. Each time, a maximum of 10 larvae were carefully collected on a disposable glass slide for observation and released in a separate glass jar to restock in the respective LRT after proper disinfection, usually a 15-second bath in 100 ppm formalin water. The stages were identified following the pictorial illustration describing the ‘prominent characteristics’ in terms of special features that appear for the first time or only at a particular stage (Nandlal and Pickering, 2005). The same approach of sampling was carried out for all the control and treatment tanks in replicates.

2.4. Post larvae (PL) conversion ratio calculation

To calculate the Post Larvae (PL) conversion ratio, the number of PL collected after 40 days of the rearing period was compared to the initial number of larvae stocked. The PL conversion ratio was determined using the following formula:

$$PLConversionratio(\%)=\frac{No.ofCollectedPL}{No.ofinitialstockedLarvae}\times 100$$

2.5. Monitoring of water quality parameters

Temperature, Dissolved oxygen (DO) and pH was monitored daily using a digital multimeter (HQ 40d portable multimeter, Hach, USA) from different tanks. Alkalinity and Total Ammonia (mg l^–1) was determined using a commercial water test kit (HANNA).

2.6. Digestive enzyme activities

Samples were prepared for digestive enzyme activities following modified protocol of Pavasovic (2004). Briefly 1 g of Larvae were collected, grinded and thoroughly washed three times in a homogenate buffer solution (10 M sodium citrate/0.1 M NaCl; pH 7.0). Finally, at ambient temperature, the homogenate samples were centrifuged at 13,000 rpm for 10 min. The supernatant was then collected and kept at −20 °C for future use.

Bernfeld's (1955) starch hydrolysis method, which measured the increase in reducing power of buffered starch solutions, was used to quantify the amylase activity. The reaction mixture contained 0.125 ml of 2% (w/v) starch solution, 0.125 ml of 0.1 M citrate phosphate buffer (pH 7.5), and 0.5 ml of enzyme source. The absorbance at 600 nm was measured in comparison to a blank after an hour of incubation at 37 °C. Immediately following the incubation period for the blank, the enzyme supply was added.

Using the casein-hydrolysis method described by Furne et al. in 2005, the protease activity was assessed. Casein was used as a substrate. The reaction mixture included 0.1 ml of supernatant (an enzyme source), 0.25 ml of 0.1 M glycine-NaOH buffer (pH 10), and 0.25 ml of casein at 1% (w/v). For one hour, the mixture was incubated at 37 °C. By adding 0.6 mL of an 8% (w/v) trichloroacetic acid solution, the reaction was stopped. It was then kept at 2 °C for an hour, centrifuged at 1800 g for 10 min, and its absorbance at 280 nm was measured in comparison to a control.

Lipase activity was assessed by measuring the breakdown of triacylglycerols, diacylglycerols, and monoacylglycerols to free fatty acids using the Bier technique (1955). Polyvinyl alcohol (1 L) was made. A 75 °C-85 °C solution of 1% polyvinyl alcohol and 5 ml of 0.1 N HCl was heated. They were then chilled and filtered. pH of the solution was adjusted to 8.0. Virgin olive oil was added to an aliquot of this solution to create 0.1 M of substrate concentration. This mixture was emulsified for 5 min. For a 4-hour incubation period at 37 °C, 1 mL of the emulsified solution, 0.5 mL of enzyme source, and 0.5 mL of phosphate-citrate buffer were added. To stop the process and separate the emulsion, 3 mL of a 1:1 mixture of ethanol and acetone was added. Phenolphthalein in ethanol was added to the reaction mixture and titrated against 0.01 N NaOH. The blanks were treated with boiling enzyme using the same method.

2.7. Superoxide dismutase activity

The SOD activity of larvae was tested using the (Hannan et al., 2024) method and the Creative BioMart, Inc., USA procedure (EC 1.15.1.1). This methodology was designed to determine the activity of superoxide dismutase in a food or enzyme sample. One g of larval sample was obtained and homogeneously mixed with 9.0 mL of deionized water for 10 min. The mixture was transferred to a centrifuge tube containing 10 mL deionized water and centrifuged at 4000 g for 15 min. The supernatant was collected and stored at −20 °C for subsequent analysis. A Tris-EDTA buffer solution (solution A) and a 0.2 mM pyrogallol solution (solution B) were prepared according to the manufacturer's instructions (EC 1.15.1.1). 2.35 mL of solution A was mixed with 2.00 mL of deionized water, then 0.15 mL of solution B was added and vortexed immediately. An aliquot was collected from the test tube and measured the absorbance at 325 nm using spectrophotometer (Peak instruments, C-7200, USA). Another aliquot was obtained after 1 min to measure the differences in absorbance between the two aliquots. Finally, the SOD activity was measured according to Wang et al. (2010) using following formula:

$$SODactivity\left(U/mg\right)=\frac{\frac{\Delta A_{325blank}-\Delta A_{325sample}}{\Delta A_{325blank}}\times 100\%}{50\%}\times 4.5\times \frac{D}{V}\times \frac{V_1}{m}$$

Where, U/mg is the SOD activity unit;

ΔA_{325 blank} is the auto-oxidation rate determined in the blank test;

ΔA_{325 sample} is the auto-oxidation rate determined using a sample;

V is the volume of sample used for testing in mL;

D is the dilution factor of the sample;

V₁ is the total volume of the sample solution in mL; m is the weight of the solid sample in gram and

4.5 is the total volume of the reaction mixture in mL.

2.8. Histological analysis

The larval sample from the control and treatment LRTs were fixed in Davidson Fixative solution for 24 h and then transfered and preserved in 70% ethanol. Histological samples were sectioned transversely, dried, and fixed in paraffin according to standard protocols (Lightner, 1996). To minimize subjectivity, hepatopancreas and muscle sections were coded and evaluated blindly using a semi-quantitative scoring system. For each group, at least five randomly selected, non-overlapping microscopic fields were assessed per section. The semi-quantitative scoring system was adapted from established aquatic histology scoring protocols (Bernet et al., 1999; Pacorig et al., 2022) , while the normalized histological integrity index was calculated in the present study as an inverse percentage transformation of the total lesion score. Hepatopancreas tissues were scored for tubular/acinar architecture disruption, tubular atrophy, acinar cell degeneration/disorganization, and necrosis/cellular debris. Muscle tissues were scored for fiber disorganization, inter-fiber separation, collagen/connective tissue disorganization, and fiber degeneration/fragmentation. Each parameter was graded from 0 to 4, where 0 indicated normal tissue and 4 indicated severe alteration, giving a total lesion score of 0–16 for each tissue.

The histological integrity index was calculated as:

$$\mathbf{Histological}\mathbf{integrity}\mathbf{index}(\%)=\left[\left(\mathbf{16}-\mathbf{total}\mathbf{lesion}\mathbf{score}\right)/\mathbf{16}\right]\times \mathbf{100}$$

2.9. Statistical analysis

Tukey's multiple comparisons test of Ordinary one-way ANOVA using GraphPad Prism (Version 9) was performed to analyse the significance among the control and treatments. Tukey's test was used to examine differences between means at a 5% probability level.

3. Results

3.1. Molting and larval development

The larvae treated with microalgae demonstrated enhanced progress in molting when compared to the control group. Furthermore, the larvae from the treatment groups showed a more homogenous growth pattern contrasting the control group, where a considerable degree of developmental stage variability was noted. Up to the sixth day of rearing (DoR), there was a notable lack of stage diversification among the larvae in both the control and treatment groups; however, larvae from the control exhibited a broad spectrum of representation across various stages during the assessments conducted at the twelfth and eighteenth DoR.

Within the given timeframe, majority of the larvae from the treatment groups (T1 and T2) progressed to the most advanced larval stage, while a contrasting trend was noted in the control group.

3.2. PL production and survival rate

Microalgae (Black algae and Spirulina powder) supplementation significantly (p < 0.05) increased the PL production (Fig. 3. C) and survival rate (Fig. 3. D) among the treatments and control group. The highest number of PL was produced in T2, followed by T1 and C. Accordingly, a maximum 93.59±2.82% survival rate was observed in T2 followed by 86.28±3.58% and 62.42±0.75% in T1 and C respectively.

Fig. 3.

A: Number of larvae l^–1 water per treatment; B: Initial number of total larvae per treatment. C: Total PL production in different treatments; D: Survival rate of PL in different treatments (After 40 days of rearing).

3.3. Water quality parameters

The average mean values of water quality parameters, such as temperature, dissolved oxygen (DO), pH, alkalinity, and total ammonia, during the experimental period under different treatments, are presented in Table 1. There were no significant differences (P > 0.05) in the temperature, pH, DO, or alkalinity among the treatments during the experimental period.

Table 1.

Water quality parameters of different treatments.

Treatments	Temp ( °C)	pH	Total Ammonia (mgL–1)	DO (mgL–1)	Alkalinity (mgL–1)
(T1)	30.55±1.07a	7.81±0.31a	0.24±0.010a	5.62±0.46a	149±3.56a
(T2)	30.48±1.10a	7.80±0.44a	0.22±0.013a	5.63±0.54a	154±6.53a
(C)	30.38±1.03a	7.79±0.34a	0.22±0.023a	5.61±0.44a	152±6.43a

3.4. Digestive enzymes activity

Microalgae supplements significantly (p < 0.05) influenced the digestive enzyme activity among the experimental groups. Amylase enzyme activity showed a significant increase among all the treatments (Fig. 4A), however, protease and lipase enzyme activity differed significantly only with the control group (Fig. 4B and C).

Fig. 4.

Digestive enzyme activity of larvae in different treatments, A. Amylase, B. Protease and C. Lipase.

3.5. Superoxide dismutase activity

In contrast to the survival rate and digestive enzyme activity, significantly higher (p < 0.05) SOD activity was recorded in the control group C, compared to T1 and T2, however, no significant difference in SOD was observed among T1 and T2 (Fig. 5).

Fig. 5.

SOD enzyme activity in different treatments.

3.6. Histological analysis

The histological images of the hepatopancreas of post-larvae reveal distinct variations in tissue morphology. In T₂, the acinar cells are well-organized, with intact lobular architecture and well-defined hepatopancreatic granules, indicative of a physiologically optimal state (Fig. 6).

Fig. 6.

Representative photomicrograph of the hepatopancreas histology of Macrobrachium rosenbergii post-larvae at 40X magnification, showing (a) T₂, (b) T₁, and (c) control groups. AC: Acinar Cells; P: parenchyma; NS: Necrosis; TA: Tubular atrophy.

In T2, the acinar cells of hepatopancreas were compact and well organized, with preserved hepatopancreatic architecture and clearly visible parenchymal structure, indicating better tissue integrity. In T1, the hepatopancreas showed moderate tissue organization, with visible acinar cells and mild irregularity in the arrangement of the parenchyma compared with T2. However, tissue damage was not evident in T1. In contrast, the control group showed marked histological alteration, including acinar cell disarray, tubular atrophy, and necrotic areas, indicating reduced hepatopancreatic integrity. Overall, the histological observations indicate that T2 showed the best tissue condition, T1 showed an intermediate but improved condition, and the control group showed the greatest structural deterioration.

The histological analysis of the muscle of post-larvae revealed (Fig. 7) distinct patterns that are indicative of various connective tissue types. T₂ (Fig. 7a) showed a dense and parallel arrangement of collagen fibers, which is characteristic of dense regular connective tissues such as tendons.

Fig. 7.

Representative photomicrographs of the muscle histology of Macrobrachium rosenbergii post-larvae with 10X magnification showing (a) T2, (b) T1, and (c) control groups, M indicates muscle fiber.

In contrast, T1 (Fig. 7b) displayed a more loosely organized collagen fiber structure, resembling that of loose connective tissue, which provides elasticity and support to adjacent structures. The control group (Fig. 7c) presented a wavy and undulating fiber pattern, typically found in tissues that undergo multidirectional mechanical stress, such as fascia.

Semi-quantitative histological scoring supported the descriptive observations of hepatopancreas and muscle tissue architecture (Table 2). In the hepatopancreas, the control group showed the highest lesion score, mainly due to tubular architectural disruption, tubular atrophy, acinar cell disorganization, and necrotic changes. In contrast, the T2 group showed the lowest lesion score and the highest histological integrity index, indicating better preservation of hepatopancreatic structure. The T1 group showed an intermediate response, with mild to moderate tissue alteration.

Table 2.

Histological scoring and condition assessment of different treatments.

Group	Hepatopancreas lesion score /16	Hepatopancreas integrity index (%)	Muscle lesion score /16	Muscle integrity index (%)	Overall interpretation
T2	2	87.50	2	87.50	Highest tissue integrity
T1	6	62.50	7	56.25	Intermediate tissue integrity
Control	12	25.00	10	37.50	Lowest tissue integrity

A similar trend was observed in muscle tissue. The control group showed greater muscle fiber disorganization, collagen/connective tissue irregularity, and inter-fiber separation, whereas the T2 group showed more compact and organized tissue architecture. The T1 group displayed moderate structural alteration. Overall, the semi-quantitative scoring indicated that the T2 treatment had the highest tissue integrity in both hepatopancreas and muscle tissues compared with the control group.

4. Discussion

4.1. Molting and larval development

This study evaluated the potential of microalgae, to be specific, Black Algae Powder (BAP) and Spirulina Powder (SP) in mass production of M. rosenbergii post larvae (PL) to overcome the mass mortality and delayed molting issues in the prawn hatcheries of Bangladesh. To the best of our knowledge, this is the first reported intervention on BAP and SP application in any commercial prawn hatchery operation. Application of BAP and SP 6 times a day (T2) showed faster growth followed by T1 (application of SP and BAP 4 times a day) and the control group C (without BAP and SP). Concentrated beta carotene in BAP, yeast and spirulina (available with the formulation) could be the reason that facilitated this remarkable growth. Considering the higher protein content, BAP and SP frequently used as dietary supplement for larval fish, shrimps and crustaceans (Bhavan et al., 2010; Palmegiano et al., 2008). BAP, on the other hand, contains special attractants that promote rapid feeding (Acua Biomar 2020). Larvae from T1 and T2 showed more uniform growth compared to the control group (Fig. 1). Several studies reported boosted growth performance with microalgae (Spirulina, Chlorella vulgaris, and Nannochloropsis sp.) along with its powder supplementation (Bhavan et al., 2010; Lober & Zeng, 2009; Maliwat et al., 2021; Panaite et al., 2023; Verni et al., 2023). Higher specific growth rate of M. rosenbergii was also reported where Tetraselmis sp. and Nannochloropsis sp. enriched copepod fed to the experimental group (Wan Rasdi et al., 2021).

Fig. 1.

Percentages of larvae at different molt stages in different treatment during the rearing period.

4.2. PL production and survival rate

Mass larval mortality within stage III-IV & VI-IX, and hindered final moulting to PL, linked to poor feeding affinity and lethargic and spiralling movement, has been a severe issue in the prawn hatcheries in Bangladesh (Alam et al., 2019; Islam et al., 2018). Similar observations have also been reported and designated as Idiopathic Muscle Necrosis (IMN), Mid-Cycle Disease (MCD), and Exuvia Entrapment Disease (EED), alternatively known as Moult Death Syndrome (MDS), suspected to be linked to nutritional deficiency and inadequate hygiene (New & Crespi, 2009). Considering the limitations, in our previous intervention (Published in Bengali as a booklet for better understanding for the hatchery operators), we optimized the standard hatchery protocol in 2019, which successfully overcame the larval mortality issues (H. M. R. Islam et al., 2019); however, the modified hatchery protocol was capable of increasing the larvae to PL conversion up to 50–60% only. To overcome PL conversion hindrance, this study assessed the effects of SP and BAP. All treatments and control larvae rearing tanks (LRT) were operated following the modified hatchery operation protocol, while SP and BAP were applied only to T1 and T2. Within the 40 days of the culture period, the maximum number of PL was harvested from T2 followed by T1 and C. Survival rate was also reported higher both in T2 (93.59±2.82%) and T1 (86.28±3.58%) compared to the control group C (62.42±0.75%) (Fig. 3D). Poultry byproduct meal-based egg custard with fish oil and artemia were reported to increase the survival from larva to PL up to 65% than the artemia only group in clear water tank culture system (Nik Sin & Shapawi, 2017). Microalgal supplementation has been effectively demonstrated to improve metamorphosis, survival, growth, immune response and pigmentation in shrimp (De Moraes et al., 2022). Microalgae, algal powder or microalgal supplements can improve larval performance by supplying high-quality protein, essential amino acids, vitamins, minerals, pigments, and other bioactive compounds that support digestion, moulting, tissue development, and stress tolerance (Saeed et al., 2025; Tang et al., 2020). Spirulina is particularly rich in bioactive pigments such as phycocyanin and carotenoids, which have been associated with improved digestive enzyme activity, antioxidant capacity, immune response, and survival in aquaculture species (García-Encinas et al., 2025; Mahendran et al., 2021; McKinley & Ozbay, 2026). The combined nutritional, physiological, and immunostimulatory effects of microalgal supplementation during larval rearing were reported to increase larval growth and survival (Jaseera et al., 2021; Ma et al., 2020). These compounds help to reduce oxidative stress during frequent moulting and metamorphosis, which are critical periods when prawn larvae are highly vulnerable to mortality (Koletti et al., 2025; Wu et al., 2026). Protein and Lipid metabolism has been reported to be directly linked to morphogenesis of Macrobrachium rosenbergii (Fu et al., 2025), which was higher in both treatments in our study than the control group. Apart from several intrinsic controls, numerous extrinsic factors viz., temperature, availability of nutrients, and water chemistry significantly influence the hormones triggering the molting process in crustaceans (Lemos & Weissman, 2021). Transferring the larvae into a separate fresh LRT at every three days during the reported rearing period offered the larvae a fresh and improved environment, which also ensured the required nutrient supply through SP and BAP supplementation, resulting in improved nutrient assimilation, enhanced antioxidant defence, and better physiological resilience of larvae receiving microalgal supplementation, which might be linked to higher survival compared to the control group (Tang et al., 2020). Moreover, this transfer process also evacuated uneaten artemia from the system, which, on the other hand, limits the risk of biomagnification and transportation of opportunistic pathogens to larvae due to continuous inputs of nutrients and fecal materials as several studies reported that Artemia-associated bacteria are not just incidental contaminants; they can be a major source of hatchery microbial load (Lee et al., 2024; Vaseeharan & Ramasamy, 2003; Villamil et al., 2003). Limited information is available on the application of dried spirulina on the growth and survival of M. rosenbergii PL; however, dried spirulina has been reported to replace up to 50% fishmeal in diet formulation without having any significant impact on growth and survival (Mimbela‐Ipanaque et al., 2025b). M. rosenbergii PL fed with 2% and 4% Chlorella reported significantly higher survival rates during pond rearing (Maliwat et al., 2021). Another experiment reported higher larval survival rate (70.8 and 63.3%) with higher algal concentrations (12.5 and 25×10⁵ cells/ml respectively) in a wet lab trial (Lober & Zeng, 2009).

4.3. Water quality parameters

Water quality remained stable throughout the experimental period, with no significant differences among the control and treatment groups. Temperature ranged from 30.38±1.03 to 30.55±1.07 °C, pH from 7.79±0.34 to 7.81±0.31, dissolved oxygen from 5.61±0.44 to 5.63±0.54 mgL^–1, alkalinity from 149±3.56 to 154±6.53 mgL^–1, and total ammonia from 0.22±0.013 to 0.24±0.010 mgL^–1. These values aligns with the reported suitable raring environments, and is comparable across treatments (Haslawati et al., 2022; Kasan et al., 2023). Stable temperature, pH, dissolved oxygen, alkalinity, and ammonia levels are essential for successful Macrobrachium rosenbergii larval development, moulting, feeding, and survival (Mallasen & Valenti, 2007). Moreover, the results suggest application of dried microalgae did not negatively affect water quality during hatchery rearing, supporting its safe use under commercial larval production conditions (Kasan et al., 2023; Mathew et al., 2021a).

4.4. Digestive enzymes activity

The nutritional physiology of crustaceans is greatly induced by the digestive enzyme activity, which can control growth, moulting cycle and complicated dietary composition either directly or indirectly (Seenivasan et al., 2016). In the current study, amylase, protease and lipase enzyme activities increased (P < 0.05) significantly in the treatment groups, T2 & T1 compared to the control group, C (Fig.4). Aligning with the observation, significantly higher activities of protease, amylase, and lipase enzyme were reported in the prawns fed with 50% of A. platensis (Radhakrishnan et al., 2016). Besides microalgal powder and periphyton-supplemented feed was also reported to increase digestive enzyme activities in Penaeus monodon (Anand et al., 2014; Tang et al., 2020).

4.5. Superoxide dismutase activity

Significantly lower Superoxide dismutase (SOD) activity was recorded both in T2 and T1 compared to C. To explain the findings, first, we need to know that the SOD enzyme is one of the crucial therapeutic agents that inhibit oxidative stress (Khan & Younus, 2025). Superoxide anions produce harmful oxygen metabolites that affect hemocyte function. These metabolites include oxygen/O₂, hydrogen peroxide/H₂O₂, ion hydroxide/OH-, protease, and reactive oxygen species (ROS) (Campa-Cordova et al. 2002). According to Rodriguez and Moullac (2000), phagocytosis, encapsulation, aggregation nodulation, melanocytes, and cytotoxicity are thought to be the mechanisms by which these reactive chemicals carry out their action and eliminate invasive infections. Hence, spirulina and dried spirulina powder, being a potent antibacterial agent (Alshuniaber et al., 2021; Hirahashi et al., 2002; Watanuki et al., 2006) and powerful immune modulator for fish and mammals (Pradhan et al., 2012), inhibited mass exposure of any infectious invasive agents to the larvae reared in the treatment tanks compared to the larvae reared with no spirulina supplementation in the control tanks resulted lowered SOD enzyme activity in the treatments relative to the control group. Consistent with the findings of the current study, enhanced immunity and increased resistance in L. vannamei against Vibrio alginolyticus was also reported in a study where heat-extracted spirulina was used as a therapeutic agent (Lin et al., 2010; Tayag et al., 2010).

4.6. Histological analysis

The histological analysis of the hepatopancreas and muscle tissues of Macrobrachium rosenbergii post-larvae demonstrated variations in tissue structure across the different treatment groups. Muscle and hepatopancreatic histology can be used to assess the health of crustaceans (Islam et al., 2024; Sun et al., 2015). To our knowledge, this study is one of the very few, if not the only, investigations to describe the histological changes in post-larval Macrobrachium rosenbergii after microalgae supplementation.

Muscle tissue analysis reinforces the role of microalgae in tissue maintenance. The dense, parallel collagen fibers observed in T₂ suggest enhanced structural development, which is typical of dense regular connective tissue. Similar results were obtained by Al-Deriny et al. (2020), who showed that Spirulina promotes collagen synthesis and supports the structural integrity of muscle fibers in fish, leading to improved mechanical properties and resilience (Al-Deriny et al., 2020). Moreover, this parallel alignment is crucial for providing tensile strength in a single direction, as supported by the findings of (Killian et al., 2012), who highlighted the role of collagen fiber orientation in the mechanical properties of tendon tissue . On the other hand, the less organized collagen structure seen in T₁ and the wavy fiber pattern in the control group highlight the dose-dependent effects of microalgae supplementation. Such findings align with studies suggesting that inappropriate frequencies of Spirulina may not sufficiently stimulate collagen synthesis or enhance tissue regeneration (Siddik et al., 2022).

Treatment T₂, which received the highest frequency of microalgae supplementation, showed well-organized acinar cells and intact lobular architecture, indicating an optimal physiological state. This structural integrity supports efficient exocrine functions, including enzyme synthesis and secretion, as well as nutrient assimilation (Hue-Beauvais et al., 2021; Ronquillo et al., 2012; Saleh et al., 2022; Wang et al., 2026). These results are consistent with findings from Mathew et al. (2021b) who found that Chlorella vulgaris improved overall larval development, providing optimal conditions for larval survival, growth, and health of the prawn larvae. (Khalila et al., 2018), however demonstrated the dietary supplementation with Spirulina platensis in Nile tilapia (Oreochromis niloticus) found significantly improved tissue integrity and growth performance by reducing cellular stress and enhancing overall cellular function.

T1, which received microalgae supplementation four times daily, showed an intermediate but clearly improved hepatopancreatic condition compared with the control group. The presence of visible acinar cells and relatively preserved parenchymal organization suggests that microalgae supplementation supported hepatopancreatic structure and function (El-Araby et al., 2022; Silva et al., 2022). However, the improvement was more pronounced in T2, indicating that a higher frequency of supplementation provided greater tissue-level benefits. Thus, the histological findings demonstrate a positive frequency-dependent response, with T1 showing partial improvement and T2 showing the highest hepatopancreatic integrity among the experimental groups. These observations are consistent with previous studies that showed the impact of enriched microalgal diets on hepatopancreatic function (Acharjee et al., 2025; Kim et al., 2026). Reduced secretion of digestive enzymes, including amylases and proteases, in response to dietary changes has been reported in prawns and other aquatic species, leading to decreased nutrient absorption capacity (Liang et al., 2022; Martínez Soler et al., 2023). Rahman and his colleagues observed that lower doses of Spirulina supplementation in fish diets might not be sufficient to achieve full tissue protection and immune enhancement, as seen in higher dosage treatments (Rahman et al., 2023).

In the control group, where no microalgae were added, significant tissue degradation was observed, including acinar cell disarray, atrophy, and necrosis. This would result in marked dysfunction in enzymatic production and absorption, severely compromising the organ’s exocrine and metabolic roles (Veerabadhran et al., 2023) .Such results also align with studies that highlight the hepatoprotective role of Spirulina, where the absence of its antioxidant properties leads to oxidative stress (Ren et al., 2021)

This study's findings suggest that the frequent application of microalgae supplements, especially in the form of Spirulina and Black Algae Powder, can significantly improve tissue integrity and growth performance in aquaculture species. The results further support the growing body of literature that emphasizes the health-promoting effects of microalgae in aquatic organisms, particularly in enhancing immune response, tissue repair, and overall resilience (Roques et al., 2022). There are no previous studies to directly compare with the current research on the histological effects of microalgae supplementation on hepatopancreas and muscle tissue in post-larval Macrobrachium rosenbergii. Future studies should investigate the molecular mechanisms underlying these phenotypic improvements by examining the expression of key genes related to ecdysis or molting, growth regulation, including members of the crustacean hyperglycemic hormone family, and immune response. Besides, future studies should also include biochemical analyses, such as HPLC or LC-MS-based carotenoid profiling, to quantify astaxanthin, β-carotene, lutein, and their retention in PL tissues. This would further clarify the relationship between microalgal pigment uptake and improved larval performance in Macrobrachium rosenbergii. Such analyses would help clarify how microalgae supplementation influences physiological development, tissue recovery, and health performance in M. rosenbergii.

5. Conclusion

On set of different diseases in the brakishwater shrimp farming shifting the farmers more into farming freshwater prawn created a massive demand of prawn PL in the recent years. Instead of the high demand, domestic prawn hatcheries could not produce required PL due to facing mass mortality during the production cycle. At these circumstances, findings of the study will help the hatcheries to ensure sustainable production of healthy PL to meet the growing demand. The study clearly revealed the health benefit of microalgae supplementation by enhanced digestive enzyme activity, lower stress level and better growth performance during the larval developmental stages with maximum survival rate. However, further study is needed to reveal the interaction of the bioactive compounds of microalgae responsible for the better performance. Further interventions are also required to investigate the expression of immune genes in relation to the bioactivity of the microalgae and fine-tune the dose of algal supplementation.

The present study concluded that the microalgal supplement in prawn hatchery could provide better survival and growth performance. A significant increase in immunity and improved growth of M. rosenbergii post-larvae reared with microalgal supplement. The outcomes of the present study will pave the way to mitigate larval mortality and reap better profit for the M. rosenbergii hatchery owners besides serving the prawn farmers and the economy of the nation.

Ethical statement

The authors were conscious of animal ethics clearance from the adhering authorities. Nevertheless, experimental protocol was permitted by Bangladesh Fisheries Research Institute (BFRI), and the research was carried out by contemplating the guidelines of the ‘Animal Welfare Act 2019′ approbated by the National Parliament of Bangladesh.

Ethics declaration

This study was conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. This study was approved by the Bangladesh Fisheries Research Institute.

CRediT authorship contribution statement

H M Rakibul Islam: Writing – review & editing, Writing – original draft, Supervision, Resources, Investigation, Formal analysis, Conceptualization. Md. Shoebul Islam: Writing – review & editing, Methodology, Formal analysis, Data curation. Md. Touhidul Islam: Methodology, Data curation. Md. Iqramul Haque: Methodology, Data curation. Mst. Aziza Begum: Writing – review & editing, Formal analysis. Mizanur Rahman Washim: Methodology, Data curation. Md. Shariful Islam: Methodology, Data curation. ASM Tanbirul Haque: Methodology, Data curation. Md. Harunor Rashid: Supervision, Resources. Md. Zulfikar Ali: Project administration.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: H M Rakibul Islam reports financial support was provided by Bangladesh Fisheries Research Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• Data will be made available on request.