Effect of fucoidan extracted from the brown seaweed, Cystoseira indica on growth, survival, immune response and disease resistance to Vibrio parahaemolyticus in Penaeus vannamei (Boone, 1931)

Introduction

Aquaculture is cultivating or rearing aquatic organisms like finfishes, molluscs, crustaceans and aquatic plants (Huntingford et al., 2012) [14] . In aquaculture, cultured shrimps are among the most economically and nutritionally important products. Due to their high acceptance in global markets, they also help developing countries earn revenue in dollar terms (Bondad-Reantaso et al., 2012) [4] . In 2020, white-leg shrimp (Penaeus vannamei) contributed 51.7% to total crustacean aquaculture with a total production of 5.8122 million tonnes (FAO, 2022). White-leg shrimp dominated the crustacean aquaculture sector because of their benefits, which include their high stocking density, high growth rate, resistance to a wide range of salinities and temperatures, and lower dietary protein needs than P. monodon (Briggs et al., 2004) [5] . Diseases have posed serious obstacles to the shrimp farming sector, making it more difficult to grow and sustain. A wide variety of pathogens, including bacteria, viruses, fungi, and parasites, can infect shrimp. The primary problem was viral diseases, namely White Spot Syndrome Virus (WSSV), which caused farmers to suffer large losses (Lightner et al., 2012) [17] . Disease outbreaks that may lead to mass mortality are the most discussed constraints in shrimp aquaculture. In 2016, the economic loss due to disease outbreaks was as high as USD 6 billion globally (Rizan et al., 2018) [22] . It was found that strains of Vibrio, for example, Vibrio parahaemolyticus, are the causative agents for the Acute Hepatopancreatic Necrosis Disease (AHPND) or Early Mortality Syndrome (EMS) that have resulted in severe economic losses in shrimp farming worldwide (Tran et al., 2013) [34] . The use of antibiotics, vaccines, and immunostimulants are the most common methods to prevent disease outbreaks (Apines-Amar and Amar, 2015) [2] . The downside of using antibiotics in aquaculture is that they disrupt aquatic microflora, cause harmful residues to build up in aquatic organisms, and contribute to the emergence of drug-resistant bacteria and pathogens (Singer et al., 2019) [24] . Vaccination also offers a potential solution for disease prevention.

Scientists have been exploring ways to strengthen shrimp immunity with a Vibrio "vaccine" since the early 1990s. The discovery of an alternative adaptive immune system in invertebrates, including shrimp, has made this approach even more promising. However, developing a vaccine that's ready for commercial use isn't a or easy process. Shrimp have a unique immune system that researchers are still working to fully understand, making vaccine development challenging (Amatul-Samahah et al., 2020) [1] . The use of natural compounds in aquaculture possesses a variety of advantages i.e. easier biodegradability, environmentally friendly, and less likely to contribute to antimicrobial resistance. The cost of the use of immunostimulants is also affordable, thus they are a potential treatment for the disease in shrimp farms (Smith et al., 2003; dos Santos Filho et al., 2023) [27,9] . Macroalgae, commonly known as seaweeds, are multicellular, photosynthetic organisms that are found in almost all aquatic environments worldwide (Dillehay et al., 2008) [8] . Due to the presence of bioactive compounds such as laminarin, fucoidan, carrageenan, and alginate, they are considered to be a potential immunostimulant for the resistance against disease in aquaculture. Fucoidan, a sulfate and L-fucose-rich polysaccharide has gained a lot of attention globally, especially in the food and pharmaceutical industries, due to its potential therapeutic applications. Its significant biological effects are attributed to its distinctive structure. Fucoidan has been known to possess antioxidant, anticancer, anticoagulant, antithrombotic, immunomodulatory, antiviral, and anti-inflammatory properties (Luthuli et al., 2019) [19] . Fucoidan was found to increase the immune response in white-leg shrimp by activation of prophenoloxidase (proPO) and haemocyte degranulation which resulted in enhanced resistance against V. alginolyticus infection. In a study by Sudaryono et al., (2018) [29] oral administration of hot water extract from Brown Seaweed, Sargassum cristaefolium was found to increase immune response in white-leg shrimp and resistance against Vibrio parahaemolyticus. Fucoidan extract from brown seaweed, Cystoseira trinodis as a feedadditive in Litopenaeus vannamei enhanced growth, immune response and disease resistance against WSSV (Salehpour et al., 2021) [23] . Moreover, the fucoidan from Sargassum wightii enriched with artemia nauplii reduced WSSV-induced mortality in Penaeus monodon postlarvae (Madasamy et al., 2012) [20] . Thus, numerous studies support the positive effect of fucoidan extract on innate immunity and disease resistance against pathogens in shrimps.

Materials and Methods

The brown seaweed Cystoseira indica was collected from the rocky shore areas along the Veraval coast in Gujarat, India. The collected seaweed was washed thoroughly and allowed them to dry in sun-shade. After drying, a mixer grinder was used to grind it and fibrous material was removed by passing the dried mixture through a mesh sieve.

A fucoidan extraction was obtained following the method described by Yang et al. (2008). A fucoidan extraction from the brown seaweed was obtained the method described by (Patel et al., 2025) [21] . The fucoidan yield was calculated based on the following formula:

Weight of obtained fucoidan (g) Weight of dried biomass (g) × 100

An experimental animal, white-leg shrimp (Penaeus vannamei) post larvae with an average weight of 0.012 g were were brought from a commercial shrimp hatchery, Gujarat. An oxygenated post larvae in polythene bags were stocked in plastic tanks of 500 L capacity and fed a commercial diet for 7 days to acclimatize them to the experimental conditions. Four experimental groups in triplicate manner were listed as follows: Control (T 0 ) (Basal diet with no fucoidan extract), T 1 (0.5% C. indica fucoidan extract), T 2 (1% C. indica fucoidan extract), and T 3 (2% C. indica fucoidan extract) with 35% protein level were formulated (Hardy 1980). A uniform sized post-larvae were stocked at 20 numbers/ tank. The experiment was carried out for 60 days were fed two times a day at the rate of 10% of body weight. The ingredients used in the formulation of different experimental diets were fishmeal, rice bran, groundnut oil cake, tapioca flour, vitamin, and mineral premix. All these were purchased from the local market. The composition of the experimental diet was given in Table 1. The required quantities of ingredients were weighed accurately, mixed, and hand-kneaded to require consistency with just a sufficient quantity of water to get smooth dough. The prepared dough was cooked under steam in an autoclave at 121 °C and 15 lbs. pressure for 10-15 minutes. The cooked feed was cooled to room temperature rapidly by spreading in an enamel tray and the required dose of fucoidan extract and vitamin-mineral premix were added, mixed, and blended. The dough was extruded through a pelletizer having a 2 mm diameter. The pellets were spread on a plastic sheet and sun-dried till the moisture content was reduced to less than 10%. Diets were packed separately in high-density polythene bags, labeled, and stored on a wooden shelf at room temperature for further use. The growth performance of shrimp was assessed in terms of weight attained, specific growth rate (SGR%/day), feed conversion ratio (FCR), protein efficiency ratio (PER) and survival rate (%) were calculated using the standard formulae El-Sayed (1999) [10] . At the end of the feeding trial, the animals were used to measure immune parameters and resistance to Vibrio [18] . A respiratory burst activity (NBT) assay was performed as described by Song and Hsieh (1994) [28] . A total haemocyte count (THC) present over the collected haemolymph was done with a microscope and a haemocyte counting Neubauer chamber.

After the end of 60 days, ten shrimps from each treatment were used for the Vibrio parahaemolyticus disease study. The bacterial strain V. parahaemolyticus was purchased from the College of Fisheries Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab. The V. parahaemolyticus was cultured on tryptic soy agar plates (TSA supplemented with 2.5% NaCl) for 24 h and the culture plates were transferred to 10 ml of tryptic soy broth (TSB supplemented with 2% NaCl) and was incubated at 25 °C for 24h as a stock culture for the experiment.

V. parahaemolyticus culture was centrifuged at 3000 rpm for 10 minutes at 4 °C. A PBS -Vibrio parahaemolyticus suspension with OD 0.8 at 620 nm which equivalent to 6.4 × 10⁸ cfu/ml. This standardized bacterial suspension was centrifuged at 3000 rpm for 10 minutes and the pellet was resuspended in 50 µl of PBS. From this, 7.81 µl was made into 50 µl using PBS to get 1× 10 8 cfu/50 µL was made into 50 µL using PBS to get 1 x 10 8 cfu/50 µL. These bacterial suspensions were serially diluted to get the required concentration of 10 8 to 10 4 cfu/50 µL. A 100 µl of bacterial suspension in a 26 gauge number syringe containing 10 4 cfu/ml PBS (LD 50 dose) injected over the second and third abdominal segment. The study was conducted by stocking ten shrimp in each tank. Then, ten shrimps from each CIF treatment were injected the bacterial suspension and the control treatment shrimps injected with the same concentration of PBS in triplicate. The susceptibility was conducted for 10 days separately and fed with a controlled diet. The mortalities were recorded down and removed from the respective tanks. The collected growth performance, immune response and diseased mortalities were analyzed statistically by using one-way analysis of variance technique (ANOVA) in all the treatments. For comparison of different treatments, Duncans's multiple range tests were used. All the data were expressed as mean ± standard error with a statistical significant value of p < 0.05.

and Discussion Throughout 60 days, the experimental days the water parameters were monitored onweekly basis. The optimum basic parameters for the growth and survival activity were as of Temperature (29.88 ± 0.21 °C), pH (7.35 ± 0.21), Dissolved oxygen (5.89 ± 0.18 mg/l) and Alkalinity (123.80 ± 1.26 mg CaCO₃/ l).

Growth and survival of Penaeus vannamei fed Cystoseira indica fucoidan extract supplemented diets

Weight Gain (WG), Feed Conversion Ratio (FCR), Specific Growth Rate (SGR) and Protein Efficiency Ratio (PER) were significant difference (p < 0.05) between the treatment groups in Table 2. There was no significant (p > 0.05) survival rate among the treatment group in Table 2. A higher weight gain (202.8% for 500mgkg -1 and 215.1% for 1000 mgkg -1 ); higher SGR (8 -7.9% day -1 ) was observed in the optimum range of 500-2000 mgkg -1 and higher survival rate was observed against Vibrio harveyii on the effectiveness of Undaria pinnatifida fucoidan extracted diet supplementation (Trifalgar et al., 2009, 2010, 2012) [33,31,32] . The efficient protein utilization was observed in the polysaccharides Macrocystis pyrifera diet supplemented in P. vannamei juveniles (Cruz-Suárez et al., 2000; Immanuel et al., 2010) [7,15] .

Immune parameters of Penaeus vannamei fed Cystoseira indica fucoidan extract supplemented diets

Experimental shrimps fed with 2% C. indica fucoidan extract were shown significantly (p < 0.05) higher phenoloxidase activity (0.192 ± 0.00 d ), higher respiratory burst activity (0.250 ± 0.00 d ) and higher haemocyte count (8.930 ± 0.02 d ) as compared to all the other treatment groups. The immune response results were listed in Table 3. A significant (p < 0.05 higher prophenoloxidase activity, respiratory burst and total haemocyte count in experimental vannamei shrimps as compared to control shrimps when challenged with WSSV test (Immanuel et al., 2012) [20] . According to Takahashi et al. (2000) [30] , incorporating lipopolysaccharides derived from Pantoea agglomerans into the diet of Marsupenaeus japonicus significantly stimulated phenoloxidase activity and phagocytosis, thereby enhancing resistance to penaeid acute viremia virus. An elevated respiratory burst activity in groupers treated with sodium alginate (20 mg kg⁻¹) or κ-carrageenan (30 mg kg⁻¹) (Cheng et al., 2008) [6] . [13,31,16,26,3,25] .

Conclusion

The dietary inclusion study of 2% fucoidan extract derived from Cystoseira indica significantly enhances the growth performance of Penaeus vannamei, as evidenced by improved weight gain, specific growth rate (SGR) and protein efficiency ratio (PER). It also strengthens immune parameters, including increased phenoloxidase activity, respiratory burst activity, and total haemocyte count. Moreover, the treatment improves feed utilization by reducing the feed conversion ratio (FCR) and lowers mortality during a Vibrio parahaemolyticus challenge. Nonetheless, survival rates across treatments showed no significant differences (p > 0.05) after 60 days of the feeding trial.

References

1. Amatul-Samahah MA, Omar WH, Ikhsan NF, Azmai MN, Zamri-Saad M, Ina-Salwany MY. Vaccination trials against Vibriosis in shrimp: a review. Aquac Rep. 2020;18:100471.
2. Apines-Amar MJ, Amar EC. Use of immunostimulants in shrimp culture: an update. In: Biotechnological advances in shrimp health management in the Philippines. 2015. p. 4571.
3. Arizo MA, Simeon EC, Layosa MJ, Mortel RM, Pineda CM, Lim JJ, et al. Crude fucoidan from Sargassum polycystum stimulates growth and immune response of Macrobrachium rosenbergii against white spot syndrome virus. Aquac Aquar Conserv Legis. 2015;8(4):535-543.
4. Bondad-Reantaso MG, Subasinghe RP, Josupeit H, Cai J, Zhou X. The role of crustacean fisheries and aquaculture in global food security: past, present and future. J Invertebr Pathol. 2012;110(2):158-165.
5. Briggs M, Funge-Smith S, Subasinghe R, Phillips M. Introductions and movement of Penaeus vannamei and Penaeus stylirostris in Asia and the Pacific. RAP Publ. 2004;15.
6. Cheng AC, Chen YY, Chen JC. Dietary administration of sodium alginate and kappa-carrageenan enhances innate immune response of brown-marbled grouper Epinephelus fuscoguttatus and resistance against Vibrio alginolyticus. Vet Immunol Immunopathol. 2008;121(3-4):206-215.
7. Cruz-Suárez E, Ricque-Marie D, Tapia-Salazar M, Guajardo-Barbosa C. Uso de harina de kelp (Macrocystis pyrifera) en alimentos para camarón. In: Cruz-Suárez E, Ricque-Marie D, Tapia-Salazar M, Olvera-Novoa MA, Civera-Cerecedo R, editors. Avances en nutrición acuícola V. 2000. p. 227-266.
8. Dillehay TD, Ramírez C, Pino M, Collins MB, Rossen J, Pino-Navarro JD. Monte Verde: seaweed, food, medicine, and the peopling of South America. Science. 2008;320(5877):784-786.
9. dos Santos Filho LG, Diniz FM, Pereira AM. Immunostimulants derived from plants and algae to increase resistance of Pacific white shrimp (Litopenaeus vannamei) against Vibriosis. Stud Nat Prod Chem. 2023;77:297-337.
10. El-Sayed AFM. Alternative dietary protein sources for farmed tilapia, Oreochromis spp. Aquaculture. 1999;179(1-4):149-168.
11. FAO. The state of world fisheries and aquaculture 2022: towards blue transformation. Rome: FAO; 2022.
12. Hardy R. Fish feed formulation. Lectures presented at FAO/UNDP Training Course in Fish Feed Technology. Seattle: University of Washington; 1980. p. 233-240.
13. Huang X, Zhou H, Zhang H. Effect of Sargassum fusiforme polysaccharide extracts on Vibriosis resistance and immune activity of shrimp Fenneropenaeus chinensis. Fish Shellfish Immunol. 2006;20(5):750-757.
14. Huntingford F, Kadri S, Jobling M. Introduction: aquaculture and behaviour. In: Aquaculture and behaviour. 2012. p. 1-35.
15. Immanuel G, Sivagnanavelmurugan M, Balasubramanian V, Palavesam A. Effect of hot-water extracts of brown seaweeds Sargassum spp. on growth and resistance to white spot syndrome virus in shrimp Penaeus monodon postlarvae. Aquac Res. 2010;41(10):1545-1553.
16. Kitikiew S, Chen JC, Putra D, Lin YC, Yeh ST, Liou CH. Corrigendum to fucoidan provokes innate immunity of white shrimp (Litopenaeus vannamei) against Vibrio alginolyticus. Fish Shellfish Immunol. 2013;34(1):280-290.
17. Lightner DV, Redman RM, Pantoja CR, Tang KF, Noble BL, Schofield P, et al. Historic emergence, impact and current status of shrimp pathogens in the Americas. J Invertebr Pathol. 2012;110(2):174-183.
18. López N, Cuzon G, Gaxiola G, Taboada G, Valenzuela M, Pascual C, et al. Physiological, nutritional and immunological role of dietary β-1,3-glucan and ascorbic acid in Litopenaeus vannamei juveniles. Aquaculture. 2003;224(1-4):223-243.
19. Luthuli S, Wu S, Cheng Y, Zheng X, Wu M, Tong H. Therapeutic effects of fucoidan: a review on recent studies. Mar Drugs. 2019;17(9):487.
20. Madasamy S, Thangapandi M, Palavesam A, Immanuel G. Antiviral effect of fucoidan from Sargassum wightii on shrimp Penaeus monodon postlarvae against white spot syndrome virus. J World Aquac Soc. 2012;43.
21. Patel JK, Parmar PV, Alwinpeter M, Bajaniya VC, Chavda VM, Parmar HV, et al. Effect of fucoidan from Sargassum johnstonii on growth, immune response and disease resistance to Vibrio parahaemolyticus in Penaeus vannamei. Int J Agric Food Sci. 2025;7(1):258-267.
22. Rizan N, Yew CY, Niknam MR, Krishnasamy J, Bhassu S, Hong GZ, et al. Electronic properties of synthetic shrimp pathogen-derived DNA Schottky diodes. Sci Rep. 2018;8(1):896.
23. Salehpour R, Biuki NA, Mohammadi M, Dashtiannasab A, Ebrahimnejad P. Dietary effect of fucoidan from Cystoseira trinodis on growth and WSSV resistance in shrimp Litopenaeus vannamei. Fish Shellfish Immunol. 2021;119:84-95.
24. Singer AC, Xu Q, Keller VD. Translating antibiotic prescribing into antibiotic resistance in the environment. PLoS One. 2019;14(9): e0221568.
25. Sinurat E, Saepudin E, Peranginangin R, Hudiyono S. Immunostimulatory activity of brown seaweed fucoidans against WSSV in shrimp Litopenaeus vannamei. J Appl Pharm Sci. 2016;6(10):82-91.
26. Sivagnanavelmurugan M, Thaddaeus BJ, Palavesam A, Immanuel G. Dietary effect of Sargassum wightii fucoidan on growth and immune resistance of Penaeus monodon. Fish Shellfish Immunol. 2014;39(2):439-449.
27. Smith VJ, Brown JH, Hauton C. Immunostimulation in crustaceans: does it really protect against infection? Fish Shellfish Immunol. 2003;15(1):71-90.
28. Song YL, Hsieh YT. Immunostimulation of tiger shrimp (Penaeus monodon) hemocytes for microbicidal substance generation. Dev Comp Immunol. 1994;18(3):201-209.
29. Sudaryono A, Chilmawati D, Susilowati T. Oral administration of Sargassum cristaefolium extract to enhance immune response of Litopenaeus vannamei against Vibrio parahaemolyticus. J World Aquac Soc. 2018;49(5):877-888.
30. Takahashi Y, Kondo M, Itami T, Honda T, Inagawa H, Nishizawa T, et al. Enhancement of disease resistance in Penaeus japonicus by oral administration of Pantoea agglomerans lipopolysaccharide. Fish Shellfish Immunol. 2000;10(5):555-558.
31. Traifalgar RF, Kira H, Tung HT, Michael FR, Laining A, Yokoyama S, et al. Influence of dietary fucoidan on growth and immunological response of juvenile Marsupenaeus japonicus. J World Aquac Soc. 2010;41:235-244.
32. Traifalgar RF, Koshio S, Ishikawa M, Serrano AE, Corre VL. Fucoidan supplementation improves Vibriosis resistance of Penaeus japonicus larvae. J Fish Aquac. 2012;3(1):33.
33. Traifalgar RF, Serrano AE, Corre V, Kira H, Tung HT, Michael FR, et al. Dietary fucoidan effects on growth and Vibriosis resistance of Penaeus monodon postlarvae. Aquac Sci. 2009;57(2):167-174.
34. Tran L, Nunan L, Redman RM, Mohney LL, Pantoja CR, Fitzsimmons K, et al. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome in penaeid shrimp. Dis Aquat Organ. 2013;105(1):45-55.