Evaluation of Dietary Fucoidan Supplementation Effects on Growth Performance and Vibriosis Resistance of Penaeus monodon Postlar vae

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1 Aquaculture Sci Evaluation of Dietary Fucoidan Supplementation Effects on Growth Performance and Vibriosis Resistance of Penaeus monodon Postlar vae Rex Ferdinand TRAIFALGAR 1, Augusto E. SERRANO 3, Valeriano CORRE 3, Haruka KIRA 2, Ha Thanh TUNG 1, Fady Raafat MICHAEL 2, Md. Abdul KADER 1, Asda LAINING 1, Saichiro YOKOYAMA 2, Manabu ISHIKAWA 2, and Shunsuke KOSHIO 2 Abstract: Two separate feeding trials were conducted with Penaeus monodon postlarvae to evaluate the effects of dietary Undaria pinnatifida fucoidan supplementation on growth performance and resistance to Vibrio harveyi infection. A semi-purified basal diet formulated to contain 49.65% protein, 9.21% lipid and 6.12% ash were incorporated with graded levels of fucoidan (0, 100, 500, 1000, 2000 mg/kg diet) and used in the feeding trials. In the first trial, each of the formulated diets was fed to triplicate groups of shrimp postlarvae (PL 15; mean weight mg) for 30 days. Results of the growth trial showed a significant enhancement of weight gain, specific growth rate, protein efficiency ratio and a lower feed conversion ratio in shrimp fed the mg fucoidan /kg diet in comparison to that of the shrimp fed the 100 mg fucoidan/kg diet and the control diet. Another 30 day feeding trial was conducted to evaluate the effects of fucoidan supplementation on the resistance of postlarvae to V. harveyi infection. Results indicated that dietary fucoidan supplementation had a significant influence on survival following V. harveyi infection. Shrimp fed the mg fucoidan/kg diet had a significantly higher survival than the shrimp fed the control diet. These results suggest that supplementation of Undaria pinnatifida fucoidan at a dose of mg/kg diet can be used to enhance growth performance and prevent or reduce mortalities in P. monodon postlarvae due to V. harveyi infection. Key words: Penaeus monodon; Growth performance; Fucoidan; Vibrio harveyi Microbiological diseases are considered a major obstacle in the sustainability and profitability of penaeid shrimp aquaculture. In recent years the industry has experienced serious economic losses due to the out breaks of pathogens that include fungi, bacteria and viruses (Vaseeharan 2003). Heavy mortalities in ponds are usually attributed to virus and bacterial out breaks, while in hatcheries the mortalities are mainly caused by fungi and bacteria. Several evidences also demonstrate that larval and postlarval stages are more susceptible to bacterial infection than the juvenile and adult stages (Jiravanichpaisal et al. 2007; Lavilla-Pitogo et al. 1990; Praytino et al. 1995; Hameed et al. 1995). Although the current knowledge on the immunological ontogeny of shrimp larvae has been limited, previous works indicate that the use of immunostimulating compounds could improve health status and resistance of shrimp larvae against microbial infections. To date, several substances of bacterial and fungal origin Received August 12, 2008: Accepted December 1, Science of Marine Resources, The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima , Japan. 2 Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Kagoshima , Japan. 3 College of Fisheries and Ocean Sciences, University of the Philippines in the Visayas, Miag-ao, Iloilo 5023, Philippines. Author to whom correspondence should be addressed: Laboratory of Aquatic Animal Nutrition, The Faculty of Fisheries, Kagoshima University, Shimoarata, Kagoshima , Japan. Tel.: ; fax: ishikawa@fish.kagoshima-u.ac.jp

2 R. F. Traifalgar, A. E. Serrano, V. Corre, H. Kira, H. T. Tung, F. R. Michael, Md. A. Kader, A. Laining, S. Yokoyama, M. Ishikawa and S. Koshio have been documented to enhance disease resistance and promote growth in several species of shrimp larvae (Rodriguez et al. 2007; Azad et al. 2005; Chang et al. 1999; Sung et al. 1994; Song and Sung 1990). Moreover, polysaccharides of algal origin such as carrageenan, laminaran, alginate and fucoidan have also been shown to exhibit immunomodulatory effects on shrimps (Yeh and Chen 2008; Cheng et al. 2005; Chotigeat et al. 2004; Martin et al. 1993). However, in contrast to microbial-derived immunostimulants such as -glucan, peptidoglycan, and lipopolysaccharide, which effects on shrimp immune response and physiology are wellestablished, information regarding the effects of seaweed polysaccharides on shrimp physiology, growth and disease resistance has been relatively few. Fucoidan is a fucose-enriched sulfated polysaccharides mainly produced by brown algae. This compound has been documented in vertebrates to elicit a wide range of biological activities such as anti-cancer, anticoagulant, antiviral and immune enhancing properties (Berteau and Mulloy 2003). The structure, sugar components and linkage patterns of algal fucoidan is complex and vary among different species of seaweeds. Previous reports also suggest that fucoidan extracted from different algal sources exhibits varying biological activities (Pereira-Dagger et al. 1999; Kiseleva et al. 2005). In shrimp, dietary administration of fucoidan from Cladosiphon okamuranus and Sargassum polycystum has been documented to improve immunological response and resistance against viral infection in juvenile M. japonicus (Takahashi et al. 1998) and P. monodon (Chotigeat et al. 2004) respectively. Although these results are promising, effects of fucoidan on the biological performance and resistance against bacterial infection in shrimp larvae have not been evaluated to date. The present work was conducted to determine the effects of dietary supplementation of fucoidan, prepared from Japanese brown algae, Undaria pinnatifida, on growth performance and resistance of Penaeus monodon postlarvae against Vibrio harveyi infection. Materials and Methods Fucoidan Fucoidan was prepared from a dried sporophyll powder of U. pinnatifida (Marui products Co., Ltd., Kitakyushu, Japan), as described previously (Mori and Nishizawa 1982). The polysaccharide preparation contains 72.0% fucoidan having a fucose and sulfate content of 40.0% and 23.5% respectively. Test diets The composition of the basal diet, which utilized casein supplemented with arginine and lysine as the protein source is shown in Table 1. All other ingredients were formulated to satisfy the nutrient requirements of Penaeus monodon (Shiau 1998). Fucoidan was incorporated to the basal diet at 100, 500, 1000 and 2000 mg/kg diet with the adjustment of cellulose contents to balance the nutrient levels of the test diets. A control group without fucoidan supplementation was also included, constituting a total of Table 1. Composition of the basal diet Ingredient g/100 g dry weight Casein Amino acid mix 1 Dextrin -starch Cholesterol Soybean lecithin Choline chloride HUFA-EE 2 Pollack liver oil Vitamin mix 3 Mineral mix 4 Attractants 5 -cellulose Activated gluten Agar Amino acid mix (g/100 g diet): L-lysine, 2.0; L-arginine-HCl, Highly unsaturated fatty acids ethyl esters; a mixture of eicosapentaenoic (20:5n-3) and docosahexaenoic acid (22:6n-3) ethyl esters (3:2, w: w; Oriental Yeast Co. Ltd., Tokyo, Japan). 3 Vitamin mix (mg/100 g): p-amino benzoic acid, 9.48; d-biotin, 0.38; Inositol, ; Niacin, 37.92; Ca-pantothenate, 56.88; Pyridoxine-HCl, 11.38; Riboflavin, 7.58; Thiamin-HCl, 3.79; l-ascorbyl-2-phosphate-mg, ; Folic acid, 0.76; Cyanocobalamine, 0.08; Menadione, 3.80; Vitamin A-palmitate, 17.85; -tocopherol, 18.96; Calciferol, Mineral mix (g/100 g diet) K 2 PO 4, 2.011; Ca(H 2 PO 4 ) 2.2H 2 O, 2.736; MgSO 4.7H 2 O, 3.05; NaH 2 PO 4.2H 2 O, Attractants (g/100 g diet): Glucosamine HCl, 0.8; Sodium succinate, 0.3; Sodium citrate, 0.3.

3 Effects of fucoidan on Penaeus monodon postlarvae five dietary treatment groups. The diets were prepared by thorough mixing of the dry ingredients with oil containing oil soluble vitamins followed by the addition of fucoidan dissolved in an adequate amount of distilled water. To the resulting moist mash, agar solution in 70 distilled water were added and mixed thoroughly. The dough was then passed through a pelletizer and the resulting spaghetti-like strings were air-dried using a convection oven (DK 400, Yamato Scientific Co., Ltd., Japan). The dried pellets were cut to appropriate size and stored at 28 until used. Growth Trial Penaeus monodon postlarvae (PL 15) were obtained from a commercial shrimp hatchery (Jamandre Hatcheries Inc., Iloilo, Philippines) and were transported to the laboratory facilities of the Institute of Aquaculture, University of the Philippines in the Visayas. The shrimp were acclimatized in laboratory conditions and maintained with the control diet (without fucoidan) for one week. After the acclimatization, shrimp were graded by size and groups of 25 shrimp with an average weight of mg were stocked into 15 glass aquaria with a capacity of 30 l. Each tank was provided with ample aeration that maintained dissolved oxygen levels at near saturation. Each experimental diet was fed to three replicate groups of shrimp for 30 days. Treatment groups were fed their respective diets at a rate of 15% body weight per day. This daily fed allocation was subdivided into three equal feedings at 9:00, 13:00 and 17:00. Fecal wastes and uneaten feeds were collected daily. Replenishment of 40% of culture water was done daily and complete water change were conducted every 10 days to maintain good water quality. Water temperature ranged from 27 29, ph from and salinity from ppt. A 12 h light: 12 h dark photoperiod was maintained throughout the trial. Shrimp were weighed every 10 days and the daily feed allocation was adjusted accordingly. After the feeding trial, shrimp were bulk weighed from each tank. Growth measured as the percentage of weight gain (WG), feed conversion ratio (FCR), specific growth rate (SGR) and protein retention (PER) were calculated as described previously (Teshima et al. 2004). Biochemical analysis Feed and carcass total protein was determined by the Kjeldahl method (Tecator Kjeltec System 1007, Foss, Denmark), crude lipid was determined using the method of Bligh and Dyer (1959). Ash and moisture contents were analyzed according to the Association of Official Analytical Chemists methods (AOAC 1990). Disease resistance trial To assess the effects of fucoidan supplementation on disease resistance, a group of 150 postlarvae were stocked in five 200 l circular fiberglass tanks and maintained with each of the experimental diets for 30 days. Feeding and experimental rearing conditions were similar to that as described above. After the 30 day feeding period, 100 postlarvae were collected from each treatment groups and subjected to a bacterial challenge test with virulent Vibrio harveyi (PN-9801), obtained from the bacterial collection of SEAFDEC Aquaculture Department, Philippines. This bacterial pathogen was previously isolated from the hepatopancreas of diseased P. monodon during the epizootics of luminescent vibriois in shrimp farms at Negros Occidental, Philippines. Pure bacterial isolate was maintained at 80 in nutrient broth (NB, BBL; Becton, Dickinson and Company, NJ, USA) supplemented with 2% NaCl and 10% glycerol. Vibrio harveyi was grown in tryptose soy broth (TSB, BBL; Becton, Dickinson and Company, NJ, USA) supplemented with 2% NaCl and in thiosulfate citrate bile salt sucrose agar media (TCBS, Difco; Becton, Dickinson and Company, NJ, USA) before use. Shrimp were infected by immersion for 6 h in an aerated viable suspension of V. harveyi at a concentration of 10 7 CFU/ml. Another group of 100 shrimp were immersed in sterilized seawater without bacteria and serve as the non-infected control group. Following the bacterial challenge, shrimps from each treatment group including

4 R. F. Traifalgar, A. E. Serrano, V. Corre, H. Kira, H. T. Tung, F. R. Michael, Md. A. Kader, A. Laining, S. Yokoyama, M. Ishikawa and S. Koshio Table 2. Survival, weight gain (WG), feed conversion ratio (FCR), specific growth rate (SGR) and protein efficiency ratio (PER) of P. monodon postlarvae fed diets supplemented with graded levels of Undaria pinnatifida fucoidan for 30 days Survival (%) WG (%) FCR SGR (%/day) PER (%) Fucoidan supplementation levels (g/100 g diet) a a a a b b a b b a b b a b b a a a a a a b b b b Values are means S.E.M. of three group of shrimp. Mean values having similar superscripts are not significantly different (P 0.05). the non-infected control were divided into triplicate groups of 25 shrimp and maintained in clean aquaria with aerated sterilized seawater at 34 ppt and 27. Mortalities were monitored daily until 20 days post-challenge and the cause of death was verified by re-isolation of the bacteria from the moribund or dying shrimp. The isolated pathogen was identified by biochemical characterization using the API 20E strips (bio-mérieux Vitek Inc.) following the manufacturer instructions. Some additional tests including Gram-stain, motility, swarming, luminescence, growth at different NaCl concentrations (0, 3, 6, 10%) and at different temperatures (4, 20, 35 ), sensitivity to ampicillin (10 g) and 0/129 (10 g), and test for the utilization of D-glucosamine and D-cellubiose as sole carbon source were performed to satisfy the required biochemical tests outlined by Alsina and Blanch (1994) for the identification of V. harveyi. Statistical analysis Data on growth and survival following a bacterial challenge test were analyzed by one way analysis of variance (ANOVA) and Tukey s honest significant difference test (super ANOVA, ver.1.11, Abacus Concepts, Berkeley, California, USA). All probability values were set at 0.05 level of significance. Results Growth Trial WG, FCR, SGR, PER and survival of shrimp after a 30-day feeding with diets supplemented with various levels of U. pinnatifida fucoidan are summarized in Table 2. WG and SGR were significantly increased with increasing dietary fucoidan supplementation levels with optimum Table 3. Proximate composition of the basal diet and whole body of P. monodon postlarvae fed with diets supplemented with graded level of Undaria pinnatifida fucoidan for 30 days Inclusion levels of Fucoidan (mg/kg diet) Crude protein (%) Lipid (%) Crude ash (%) Basal diet whole body Values are the means of triplicate group S.E and expressed as percent of the dry matter. growth response obtained at mg fucoidan/kg diet. Lowest growth was obtained in the control treatment that was not significantly different from the lowest level of supplementation (100 mg fucoidan/kg diet). Survival of all the treatment groups was high (86 92%) and not affected by fucoidan supplementation levels. Nutrient utilization parameters such as FCR and PER were also significantly enhanced by fucoidan supplementation and exhibited a similar trend to that of the WG and SGR. Fucoidan supplementation levels had no prominent effects on body composition. No significant differences in body protein, lipid, and ash contents were observed among the treatments (Table 3). Disease resistance trial Supplementation levels of fucoidan had a significant effect on survival following V. harveyi challenge (Fig. 1). Survival in all the treatment groups fed diets containing fucoidan was higher than in the control group. Survival was lowest in the control group and increased with

5 Effects of fucoidan on Penaeus monodon postlarvae Survival (%) a Control d c Fucoidan levels (mg/kg diet) Fig. 1. Survival of P. monodon postlarvae 20 days after an immersion challenge with V. harveyi. Five triplicate groups of 25 postlarvae were fed diets containing graded levels of U. pinnatifida fucoidan (0, 100, 500, 1000, 2000 mg/kg diet) for 30 days and challenged with V. harveyi by immersion. Represents the non-infected control group. The mean values of the bars bearing same superscript do not vary significantly (P 0.05). increasing dietary levels of fucoidan supplementation from 100 to 500 mg/kg. Higher supplementation levels at 1000 and 2000 mg/kg had no additional benefits in terms of survival and were comparable to that of 500 mg/kg diet. Luminescent bacterial colonies identified as V. harveyi were isolated from the moribund shrimp. Discussion The efficacy of fucoidan as an immunostimulatory factor in higher vertebrates is well documented. Mitogenic activation of Bursa-cells, B-lymphocytes, induction of cytokine (tumor necrosis factor alpha) release, enhancement of macrophage phagocytic activity and activation of natural killer cells are some of the prominent immunological effects of fucoidan in higher vertebrates (Heinzelmann et al. 1998; Berteau and Mulloy 2003; Hayashi et al. 2008). Furthermore, recent works indicate that this compound is also effective in enhancing immunological response and viral resistance of juvenile shrimp (Takahashi et al. 1998; Chotigeat et al. 2004). However, these previous studies were conducted b b b in short durations and effects of fucoidan on growth have not been determined to date. Moreover, bacterial infection is a serious problem in shrimp larval rearing but no studies have been conducted about the effects of fucoidan on bacterial resistance in shrimp larvae. To the best of our knowledge the present study is the first to document the effects of dietary fucoidan supplementation on growth performance and enhancement of bacterial resistance in shrimp postlarvae. In the present study, survival of shrimp after the feeding trial was not affected by dietary fucoidan supplementation but WG, FCR, SGR and PER were significantly improved. Enhanced growth performance is apparent at 500 mg/ kg supplementation with no additional growth benefits observed at higher supplementation levels up to 2000 mg/ kg, indicating that 500 mg/kg supplementation is adequate in promoting growth in this species. This finding is consistent with previous reports, indicating the growth promoting benefits of immunostimulant application in shrimp. Growth enhancement was also observed in P. japonicus fed peptidoglycan-supplemented diets (Itami et al. 1998), in P. monodon larvae fed diets with Vibrio vulnificus bacterin (Song and Sung 1990) and in juvenile Litopenaeus vannamei fed diets supplemented with Ergosan, a commercial product prepared from a brown alga, Laminaria digitata (Montero-Rocha et al. 2006). Enhancement of nutrient digestibility, resulting in efficient protein utilization and improvement of growth rate has also been reported in juvenile L. vannamei fed diets supplemented with polysaccharide from Macrocytis pyrifera (Cruz-Suarez et al. 2000). Despite these reports, the mode by which these compounds promote growth in crustacean is not yet fully elucidated. It has been suggested that growth enhancement effects of dietary immunostimulants might be attributed to the efficient nutrient digestion and assimilation caused by the activation of fixed phagocytes in the hepatopancreas that are known to produce lytic enzymes upon stimulation (Azad et al. 2005). Health improvement as a consequence of active immunological

6 R. F. Traifalgar, A. E. Serrano, V. Corre, H. Kira, H. T. Tung, F. R. Michael, Md. A. Kader, A. Laining, S. Yokoyama, M. Ishikawa and S. Koshio defense and reduced stress from opportunistic microbial pathogens has also been hypothesized to attribute for the enhancement of survival and growth of immunostimulated animals (Sung et al. 1994; Itami et al. 1989). Luminous Vibriosis disease caused by Vibrio harveyi is a serious problem associated with production losses in larval-rearing of P. monodon (Karunasagar et al. 1994; Lavilla- Pitogo et al. 1990). In recent decades, a number of studies have indicated that enhancement of immunocompetence by the use of immunostimulants appear to be a promising prophylactic approach in controlling bacterial diseases in shrimp culture (Sakai 1991; Raa et al. 1992). In the present study, all treatment groups receiving dietary fucoidan supplementation exhibited a significantly higher survival after a challenge with V. harveyi as compared to the control treatment. Promotion of a protective response is evident at 100 mg/kg supplementation with optimum response at 500 mg/kg supplementation. Higher supplementation levels ( mg/kg) have no apparent detrimental effects on survival and are statistically similar to that of 500 mg/kg supplementation. The high survival conferred by dietary fucoidan supplementation is not surprising. This compound has been known to be a potent activator of cellular immune response in mammalian models. Evidence suggests that fucoidan augments disease resistance by acting on the reticuloendothelial component of the immune system (Itoh et al. 1993; Heinzelmann et al. 1998). In shrimp, previous reports suggest that dietary fucoidan supplementation can enhance the resistance of juvenile M. japonicus (Takahashi et al. 1998) and P. monodon (Chotigeat et al. 2004) against white spot syndrome virus infection. The authors attributed these protective effects to the enhancement of hemocyte phagocytic activity and to the inhibition of viral adsorption to the host. Furthermore, Deachamag et al. (2006) elucidated that the mode by which fucoidan enhance cellular immune response in shrimp is through the activation of a phagocytosis activating protein, known to initiate and enhance hemocyte phagocytic activity. Although shrimp immunological response was not measured in this study due to the technical limitations in extracting haemolymph from the larvae, it is conceivable that the enhancement of survival after the challenge may have been due to the activation of cellular immune response, similar to that observe in juvenile shrimp. Nevertheless, the present findings show that dietary fucoidan supplementation can promote growth and enhance resistance of P. monodon postlarvae against V. harveyi infection. With the increasing restrictions on the use of antibiotics in aquaculture, utilization of fucoidan as a dietary supplement can be a promising immuno-prophylactic approach in infectious disease management in shrimp aquaculture. However, the cellular mechanisms involved on how this compound promotes growth and enhances resistance against bacterial infection in shrimp is yet to be clarified and warrants further investigations. Acknowledgement This study was supported by a research grant from the Development Program for New Bioindustry Initiative of Japan. The scholarship and support of Monbukagakusho, Japan for this work is also gratefully acknowledged. References Alsina, M. and A. R. Blanch (1994) A set of biochemical identification of environmental Vibrio species. J. Appl. Bacteriol., 76, AOAC (Association of Official Analytical Chemists International) (1990) Official methods of analysis, 15th edition. AOAC International, Gaithersburg, Maryland, USA, 1298pp. Azad, I.S., A. Panigrahi, C. Gopal, S. Paulpandi, S. Paulpandi, C. Mahima and P. Ravichandran (2005) Routes of immunostimulation vis-à-vis survival and growth of Penaeus monodon post larvae. Aquaculture, 248, Berteau, O. and B. Mulloy (2003) Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide. Glycobiology, 13, Bligh, E. G. and W. J. Dyer (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37,

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8 R. F. Traifalgar, A. E. Serrano, V. Corre, H. Kira, H. T. Tung, F. R. Michael, Md. A. Kader, A. Laining, S. Yokoyama, M. Ishikawa and S. Koshio Biotechnology (ed. By Flegel T. W.), National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand, pp Teshima, S., M. Ishikawa, M. D. Alam, S. Koshio, and F. R. Michael (2004) Supplemental effects and metabolic fate of crystalline arginine in juvenile shrimp Marsupenaeus japonicus. Comp. Biochem. Physiol. B, 137, Vaseeharan, B. and P. Ramasamy (2003). Abundance of potential pathogenic micro-organisms in Penaeus monodon larvae rearing systems in India. Microbiol. Res., 158, Yeh, S.-T. and J.-C. Chen (2008). Immunomodulation by carrageenans in the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Aquaculture, 276, Rex Ferdinand TRAIFALGAR Augusto E. SERRANO Valeriano CORRE Ha Thanh TUNG Fady Raafat MICHAEL Md. Abdul KADER Asda LAINING PL Vibrio harveyi mg/kg PL mg/kg 100 mg/kg V. harveyi 500 mg/kg 500 mg/kg