Penaeus monodon (Fabricius)

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1 Effect of vitamin C and astaxanthin on stress and disease resistance of postlarval tiger shrimp, Penaeus monodon (Fabricius) G Merchie 1,3, E Kontara 1, P Lavens 1, R Robles 1, K Kurmaly 2,4 & P Sorgeloos 1 1 Laboratory of Aquaculture and Artemia Reference Centre, University of Ghent, Gent, Belgium 2 Rovithai Ltd, Bangchak Prakanong, Bangkok, Thailand 3 Present address: INVE (Thailand) Ltd, 445 Sanambin Road, Tambon Naimuang, Amphur Muang, Phitsanulok 65000, Thailand 4 Present address: Roche Vietnam, 101 Vo Thi Sau Street, 3rd District, Ho Chi Minh City, Vietnam Correspondence: P Lavens, Laboratory of Aquaculture and Artemia Reference Centre, University of Ghent, Rozier 44, B-9000 Gent, Belgium Abstract Postlarvae of tiger shrimp, Penaeus monodon (Fabricius), were fed semipurified diets supplemented with various levels of astaxanthin (AX) and ascorbic acid-polyphosphate (ApP): three groups were fed 230 mg AX kg 1 diet combined with 100, 1700 and 3400 mg ascorbic acid (AA) kg 1 diet, respectively; two diets contained 810 mg AX kg 1 mixed with 200 and 1700 mg AA kg 1, respectively. Each treatment was run in four replicates. Incorporated levels of AA and AX, production output, and physiological condition were recorded after 4 weeks of feeding. Whole-body AA (21 47 µg g 1 ) and AX concentrations (19 35 µg g 1 ) were linked to dietary ApP and AX supply, respectively, although not significantly for the latter. The biomass of the group receiving the lower dietary ApP AX combination was significantly lower than for all other treatments, i.e. 3.1 versus 3.9 g, respectively. In the groups fed 230 mg AX kg 1 diet, significant differences in stress resistance were observed according to the dietary ApP level, i.e. raising the vitamin C content in the feed from 100 to 3400 mg AA kg 1 resulted in a concomitant drop in mortality after an osmotic shock. For the treatments receiving 810 mg AX kg 1 diet, the beneficial effect of extra dietary vitamin C was not significant. An increase in the dietary AX for shrimp fed comparable ApP levels resulted in a significant drop of the stress index from 56 to 33 (cumulative mortality index). An increased resistance to salinity shock was demonstrated in association with supplementation of high dietary AA or AX levels. No conclusive results regarding possible improved disease resistance could be made since no mortality was observed after a disease challenge with Vibrio harveyi. Introduction In view of the disease problems which are currently faced by the shrimp farming sector, management strategies have been postulated that recommend, among other things, an assessment of the quality of the postlarvae to be stocked in grow-out ponds. In this respect, the significance of an adequate diet in maintaining the health of aquaculture organisms is widely recognized. Nutritional status is considered one of the important factors which determines the ability of the animal to withstand infections. More specifically, carotenoids, vitamin E and vitamin C play important roles in animal health as antioxidants by inactivating damaging free radicals produced through normal cellular activity and from various stressors (Chew 1995). It has been suggested that the antioxidant function of these micronutrients could enhance immunity by preserving the functional and structural integrity of important immune cells. In this respect, the need for specific 1998 Blackwell Science Ltd. 579

2 Ascorbic acid and astaxanthin in tiger shrimp G Merchie et al. Aquaculture Research, 1998, 29, nutrients may be increased during infections, which could require the feeding of diets formulated for optimal immunocompetence rather than growth and survival. Lightner, Colvin, Brand & Danald (1977) first demonstrated the essential role of dietary vitamin C in preventing the development of the black death syndrome in penaeid shrimp. Recommended dietary ascorbic acid (AA) levels for shrimp using ascorbic acid-polyphosphate (ApP) are 20 and mg AA kg 1 for the postlarvae of tiger shrimp, Penaeus monodon (Fabricius), and white shrimp, Penaeus vannamei (Boone), respectively (He & Lawrence 1993; Lavens, Merchie, Ramos, Leon-Hing Kujan, Van Hauwaert, Pedrazzoli, Nelis & De Leenheer 1998). Furthermore, a significant effect was was shown to be produced on stress sensitivity for both species by high dietary AA levels, and moreover, P. vannamei displayed a higher resistance to Vibrio harveyi infection (Kontara, Merchie, Lavens, Robles, Nelis, De Leenheer & Sorgeloos 1997; Merchie et al. 1997). Crustaceans are completely dependent upon an exogenous source of carotenoids for pigmentation and various authors have suggested other vital functions of these compounds in aquatic animals (D Abramo & Conklin 1995). Shrimp do convert various carotenoids into astaxanthin (AX), but the direct supply of AX ensures more effective absorption from the diet and deposition in tissues (Menasveta, Choosuwan, Bühler, Schielre & Latscha 1994). The survival and growth of postlarval P. monodon increase according to dietary AX up to supplementation levels of 60 and 300 mg kg 1, respectively (Thongrod, Tansutapanich & Torissen 1995), while optimal pigmentation of juveniles is obtained by adding mg kg 1 of AX (Menasveta, Worawattanamateekul, Latscha & Clark 1993; Menasveta et al. 1994). Bendich (1989) reported the enhancement of both nonspecific and specific immune functions by carotenoids. In the present study, the effect of high dietary doses of vitamin C and AX on the physiological condition and disease resistance of postlarval P. monodon was assessed. Materials and methods Diet preparation Five practical diets (A E) with varying levels of ApP (Stay-C 25%, Roche, Bangkok, Thailand) and AX Table 1 Ingredient composition of the basal diet [modified from Camara et al. (1995)] Ingredient Casein 1 35 Fish meal 2 5 Squid meal 2 5 Shrimp meal 2 5 Arginine 3 1 Sucrose 4 5 Cellulose Starch 6 10 Mineral mix 7 10 Vitamin mix 7 2 Vitamin E K-carrageenan 9 4 Cholesterol Fish oil FO Soybean oil 12 5 Deoiled soybean lecithin Butylated hydroxytoluene Butylated hydroxyanisole Ethoxyquin Attractant 2 2 Percentage of diet 1 Sigma Chemical Co., St Louis, MO, USA. 2 INVE Aquaculture N.V., Baasrode, Belgium. 3 Sigma A-5131, Sigma N.V., Bornem, Belgium. 4 Sigma S Sigma C Sigma S Teshima et al. (1982), Sigma Chemical Co. 8 dl-α-tocopherol-acetate, Federa N.V., Deinze, Belgium. 9 Sigma C Sigma C Ethyl ester concentrate containing 523 mg n-3 HUFA g 1, 301 mg EPA g 1, 189 mg DHA g 1, DHA:EPA ratio of 0.62, INVE Aquaculture N.V. 12 Vandemoortele N.V., Izegem, Belgium. 13 EMULPUR N, Lucas Meyer GmbH & Co., Germany. 14 Federa N.V. 15 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinolin, Sigma E (Carophyll Pink 8%, Roche) were prepared according to Camara, Coutteau & Sorgeloos (1995) using the basal diet formula presented in Table 1. The various concentrations of ApP and AX (Table 2) were balanced by proportional quantities of cellulose. The feed ingredients were bound in a matrix using Φ-carrageenan and pelleted. The pellets were dried at 25 C for 2 days to µ5% moisture content. The dried strands were crumbled and sieved to the desired particle size ( µm). Prepared diets Blackwell Science Ltd, Aquaculture Research, 29,

3 Ascorbic acid and astaxanthin in tiger shrimp G Merchie et al. Table 2 Detected levels of L-ascorbyl-2-polyphosphate (ApP) and astaxanthin (AX) in the micro-bound diets* Diet ApP (mg AA kg 1 ) AX (mg kg 1 ) A a 230 B c 220 C d 250 D b 820 E c 800 *The AX and ApP data are means ( SD) of a single and two replicate analyses (AA: ascorbic acid). Values with the same superscript letter are not significantly different (P 0.05). were stored at 20 C during the experiment. The actual levels of ApP in the diets after pelletizing and leaching (for 10 min) were analytically verified by phosphatase digestion following the procedure for AA determination (Nelis, Merchie, Lavens, Sorgeloos & De Leenheer 1997). Analysis of AX was performed by Roche, Basel, Switzerland, using a highperformance liquid chromatograph (HPLC) assay (Weber 1992). Experimental set-up The feeding experiment was set up as a randomized block designed with four blocks each containing one replicate of five treatments. Each block consisted of five aquaria (useful volume of 30 L) connected to a gravel-bed biological filter (120 L), over which the sea water was continuously recirculated, and a water reservoir (80 L). Filtered water from the reservoir was circulated by an electric pump (1 L min 1 ) through a constant head tube to the rearing units. During the experiment, water temperature and salinity were maintained at C and gL 1. NH 4 N and NO 2 N were monitored daily and never exceeded 0.16 and 0.06 mg L 1. Illumination was provided by dimmed TL-lamps (140 lux at the water surface) for 12 h day 1. Penaeus monodon postlarvae (PL-10 stage, average individual dry weight 1.28 mg) were obtained from the Anajak Hatchery, Chonburi, Thailand, and acclimatized for 2 days prior to stocking at a density of 4 postlarvae L 1. The postlarvae were fed ad libitum four times a day (at 0800, 1200, 1500 and 1900 h) with particles of µm during the first week of feeding and µm afterwards. The tanks were siphoned twice a day before the 0800 and 1500 h feedings. Approximately 10% of the water in each biofilter was renewed daily; the salinity was adjusted with tap water to compensate for evaporation. The feeding experiment was terminated after 4 weeks of feeding (PL-40 stage). Evaluation The dietary treatments were evaluated at the end of the feeding trial based on production (survival, growth and biomass), stress and disease resistance, and incorporation of AA and AX in the shrimp. Final survival (%) was calculated by counting the remaining postlarvae in each aquarium. Individual dry weights were calculated on triplicate samples of 10 postlarvae tank 1 dried for 24 h at 60 C. A stress test was performed by means of a salinity shock following the method of Tackaert, Abelin, Léger & Sorgeloos (1989). Ten postlarvae per tank were immersed in fresh water and the survival rate was monitored at 5-min intervals over a 1-h period. The physiological condition of the animals was expressed as the cumulative stress index (CSI), calculated as the average value of the replicated treatments obtained after the individual addition of the cumulative mortalities in the consequent time intervals (Dhert, Lavens & Sorgeloos 1992). The susceptibility to a Vibrio harveyi infection was assessed applying a challenge test in which two replicates of 10 postlarvae per treatment were injected with bacterial suspension. A potentially pathogenic V. harveyi strain (STD3 70, challenge), a non-pathogenic V. harveyi strain (STD3 69, positive control) and saline solutions (NaCl 1.5% w/v, negative control) were used in the trial. The two V. harveyi strains were obtained from the collection of the Laboratory of Microbiology, University of Ghent, Gent, Belgium. A fresh batch of bacteria was taken from storage ( 80 C) and checked for purity on ZoBell agar (Difco, Bierbeck, Belgium) plates, and then grown for 24 h at 25 C on the same medium. The cells were harvested using sterile swabs and suspended in sterile solution (NaCl 1.5% w/v). Concentration was adjusted to 10 8 colony forming units (CFUs) ml 1 by measuring optical density at 550 nm. The experimental bacterial suspension, with a concentration of CFUs ml 1, was obtained by serial dilution in sterile saline solution. Experimental animals were injected 1998 Blackwell Science Ltd, Aquaculture Research, 29,

4 Ascorbic acid and astaxanthin in tiger shrimp G Merchie et al. Aquaculture Research, 1998, 29, with µl bacterial suspension ( CFUs shrimp 1 ). Mortality was recorded daily for a 5-day period. Control and challenged animals were kept in 5-L aquaria under the same rearing conditions. Samples from the challenged shrimp (hepatopancreas) were cultured on thiosulphate citrate bile-salt sucrose medium (TCBS, Difco), and various Vibrio colonies were isolated and purified for further identification in order to verify the presence of injected strains in the organ. Identification and characterization of the isolates were performed using a Biolog TM GN microplates system (Biolog Inc., Hayward, CA, USA; Garland & Mills 1991). The analysis of AX was performed by Roche, Basel, Switzerland, applying a HPLC assay (Weber 1992). The AA levels were determined by HPLC and electrochemical detection following Nelis et al. (1997). Statistical analysis The values in the tables represent the means and the statistical significance of differences in means, and were determined using a one-way analysis of variance (ANOVA) and Tukey s multiple range test (P 0.05). The homoscedasticity of variances was controlled by Barlett s test. In the case of nonhomogeneity of variances, transformations (arcsin or log) were applied. Results The ApP and AX levels included in the various diets after micro-binding are summarized in Table 2. Two groups of diets (A, B and C on the one hand, and D and E on the other) contained different levels of AX, averaging µ230 and 810 mg kg 1, respectively. The ApP levels in diets B and E did not differ significantly and varied around a mean value of 1700 mg AA kg 1. The postlarvae initially contained µg AA g 1 dry weight (DW). The whole-body AA and AX concentrations at the end of the trial are presented in Table 3. The AA concentrations varied between 114 and 252 µg g 1 DW [DW 18.8% wet weight (WW)] according to the dietary ApP supply. Shrimp fed 230 and 810 mg AX kg 1 diet contained and µg AXg 1 DW, respectively, after 4 weeks of feeding (not significantly different). The individual DW, survival and tank biomass are Table 3 Incorporation of ascorbic acid (AA) and astaxanthin (AX) in Penaeus monodon postlarvae fed diets supplemented with varying levels of vitamin C and AX over 30 days (µg g 1 dry weight; dry weight 18.8% wet weight)* Diet AX230 AX810 AA AA a AA b AA c b,c AA d AX AA AA AA AA *Data for AA are means ( SD) of four replicate analyses. Values with different superscript letters are significantly different (P 0.05). Data for AX are values of a single analysis. mg AX kg 1 diet mg AA kg 1 diet. summarized in Table 4. The growth and production output (biomass tank 1 ) of the group receiving the lower dietary ApP AX combination were significantly less than for all other treatments: 6.5 versus 7.8 mg DW and 3.1 versus 3.9 g, respectively. The highest survival occurred in the group fed diet E. In the groups fed 230 mg AX kg 1 diet, significant differences in stress resistance were observed according to the dietary ApP level (Table 5), i.e. raising the vitamin C content in the feed from 100 to 3400 mg AA kg 1, and consequently, in the body tissue from 114 to 252 µg AAg 1 DW, resulted in a concomitant drop in mortality after the osmotic shock. For treatments receiving 810 mg AX kg 1 diet, the influence of extra dietary vitamin C was less pronounced since incorporated levels were not significantly different. An increase in the dietary AX for shrimp fed comparable ApP levels (diets B and E) resulted in a significant drop of the CSI from 56 to 33. No mortality was recorded after disease induction. Therefore, the shrimp were challenged a second time using the described methodology; again no mortality occurred. Only the V. harveyi strain (STD3 70) used as a potential pathogen could be recovered from the hepatopancreas samples Blackwell Science Ltd, Aquaculture Research, 29,

5 Ascorbic acid and astaxanthin in tiger shrimp G Merchie et al. Table 4 Mean ( SD) individual dry weight (DW), survival and biomass (g tank 1 ) of Penaeus monodon postlarvae fed diets supplemented with varying levels of ascorbic acid (AA) and astaxanthin (AX) over 30 days* Diet AX230 AX810 DW (mg) AA b AA a AA a a AA a Survival (%) AA b AA a,b AA a,b a AA a,b Biomass (g) AA b AA a AA a a AA a *Values with different superscript letters are significantly different (P 0.05). mg AX kg 1 diet. mg AA kg 1 diet. Table 5 Mean ( SD) stress sensitivity (0, 2 h) of Penaeus monodon postlarvae fed diets supplemented with varying levels of ascorbic acid (AA) and astaxanthin (AX) over 30 days* Cumulative stress index ( ) AX230 AX810 AA c AA a,b AA b a AA a *Values with different superscript letters are significantly different (P 0.05). mg AX kg 1 diet. mg AA kg 1 diet. Discussion Ascorbic acid-polyphosphate has shown AA activity in both shrimp (He & Lawrence 1993; Chen & Chang 1994) and fish (Wilson, Poe & Robinson 1989; Li, Johnson & Robinson 1993), and was chosen as the AA source because of its stability (Grant, Seib, Liao & Corpron 1989). Whole-body AA concentrations reflected the vitamin C levels offered through the diet: µg AAg 1 DW or µg g 1 when expressed on a WW basis. Since the dry matter content of shrimp was not significantly different among the various treatments (18.8% DW), calculations based on WW did not increase the variability of the results. These numbers are comparable to values reported for early postlarval P. vannamei by Kontara et al. (1997): 13 and 37 µg AA g 1 WW in shrimp offered 70 and 1500 mg AA kg 1 diet (as ApP), respectively. He & Lawrence (1993) obtained 13 µg AAin1-gP. vannamei when feeding 200 mg AA kg 1 diet, which was half of the concentration (30 µg AA g 1 WW) found in the present study for equivalent treatment D. Most wild species of penaeid shrimp farmed in commercial operations contain levels of carotenoids totalling µg g 1 body weight (D Abramo & Conklin 1995). When the total carotenoid content falls below µ20 µg g 1, intensively reared P. monodon exhibit an unnatural blue colour which is a manifestation of a dietary carotenoid deficiency (Howell & Matthews 1991). The AX concentrations in the present study exceeded 20 µg g 1 WW for all groups: 22 and 31 µg g 1 for shrimp fed 230 and 810 mg AX kg 1 diet, respectively. Thongrod et al. (1995) detected µ26 µg AX g 1 in postlarval P. monodon fed 230 mg AX kg 1. The group fed the low dietary ApP AX combination yielded a significantly lower biomass than all other treatments. The dietary AA requirements reported for postlarval and juvenile P. monodon vary between 20 and 200 mg AA kg 1, supplied as ApP (Chen & Chang 1994). For AX, growth and survival are positively correlated to AX supplementations up to 300 mg kg 1 (Thongrod et al. 1995). An increased resistance to salinity shock was demonstrated in correlation with increased concentrations of AA or AX in the body tissue through supplementation of high dietary AA or AX levels. To the present authors knowledge, this is the first report of enhanced stress resistance in penaeid shrimp postlarvae related to an increase in dietary and body AX, as is shown for the AA1700 diet. The positive effect of extra AA on shrimp capacity to withstand a drop in salinity has been described before, both for P. monodon (G. Merchie et al., unpublished results) and P. vannamei postlarvae (Kontara et al. 1997; Lavens et al. 1998) fed 200 and 1500 mg AA kg 1 diet, respectively. Since this 1998 Blackwell Science Ltd, Aquaculture Research, 29,

6 Ascorbic acid and astaxanthin in tiger shrimp G Merchie et al. Aquaculture Research, 1998, 29, biological response is related to an increase in incorporated AA, it supports the hypothesis that stress creates increased AA requirements for larval fish and crustaceans (Dabrowski 1992). For the groups fed 810 mg AX kg 1 diet, no significant increase in stress resistance was noticed with elevated dietary ApP. This could be linked to the non-significant raise in AA incorporation in the shrimp body. No mortality occurred in two consecutive challenge tests, although both strains were identified as shrimp pathogens since they were luminescent, and had small and large plasmids (Prayitno 1994). The V. harveyi strain STD3 70 was isolated from diseased shrimp larvae in Indonesia and used in several pathogenicity experiments, revealing pathogenic effects for larval stages of P. monodon (Prayitno 1994). The lack of pathogenicity of the V. harveyi STD3 70 in the present experiment is unclear and could have been caused by several factors (e.g. a change in plasmid profiling, host specificity or prolonged storage time). As was expected, the V. harveyi strain STD3 69, used as a positive control to demonstrate that the mortalities obtained in the challenge trial were caused by the pathogenic characteristics of the strain and not as a result of the amount of bacteria injected, did not show any negative effect on the shrimp. Nevertheless, several reports have shown the involvement of vitamin C and AX in the immune system. Kontara et al. (1997) demonstrated that increased mortality caused by a V. harveyi infection of P. vannamei postlarvae is affected by the AA dietary supplementation level. Only the highest level of dietary AA (1500 mg kg 1 diet) appeared to be sufficient to obtain an optimal resistance to the bacterial infection. Also, Kanazawa (1997) reported that vitamin C was effective in increasing resistance of kuruma shrimp, P. japonicus (Bate), when the Vibrio sp. NJB strain (isolated from diseased P. japonicus) was introduced. One week after inoculation, the survival rate of shrimp fed diets containing 50 mg AA kg 1 (as ApP) was 80%, while in the control (ApP-free), only 14% survived. In the past, research on the function of carotenoids on immunity and health has been focused on β- carotene, and more recently, it has been concentrated on canthaxanthin and astaxanthin (Chew 1995). Bendich & Shapiro (1986) suggested that the mode of action is either a retinoid effect, a conversion to a compound with retinoid effect, a free radical scavenging activity or a combination of the above. The development of a standard challenge methodology for shrimp should verify if the observations which have been made for humans and rats (Bendich & Shapiro 1986) are also valid for crustaceans. Acknowledgments This study was supported by Rovithai Ltd, the European Union (project TS3*-CT ) and the Concerted Actions programme of the Ministry of Science. Endhay K. M. Kontara acknowledges a Ph.D. grant from the Belgian Administration for Development Cooperation. Astaxanthin analyses were performed at the Department of Vitamin Research and Development (Dr Joseph Schielre, Roche, Basel, Switzerland). The authors wish to thank Dr Hans Nelis for supervising the ascorbic acid analyses, Hilde Van Duffel for supervising the challenge test, Marina Baptista and Pascale Hoste for technical assistance, and Drs Yew-Hu Chien and Brian Hunter for critically reading the manuscript. References Bendich A. (1989) Carotenoids and the immune response. Journal of Nutrition 119, Bendich A. & Shapiro S.S. (1986) Effect of β-carotene and canthaxanthin on the immune responses of the rat. Journal of Nutrition 116, Camara M. R., Coutteau P. & Sorgeloos P. (1995) Effect of dietary phosphatidylcholine on growth and lipid composition of postlarval Penaeus vannamei (Boone). In: Memorias II Congreso Ecuatoriano de Acuicultura (ed. by J. Calderón & P. Sorgeloos), pp Escuela Superior Politécnica del Litoral, Guayaquil. Chen H.Y. & Chang C.F. (1994) Quantification of vitamin C requirements for juvenile shrimp (Penaeus monodon) using polyphosphorylated l-ascorbic acid. Journal of Nutrition 124, Chew B.P. (1995) Antioxidant vitamins affect food animal immunity and health. Journal of Nutrition 125, 1804S- 1808S. D Abramo L.R. & Conklin D.E. (1995) New developments in the understanding of the nutrition of penaeid and caridean species of shrimp. In: Swimming Through Troubled Water, Proceedings of the Special Session on Shrimp Farming, Aquaculture 95 (ed. by C. L. Browdy & J. S. Hopkins), pp World Aquaculture Society, Baton Rouge, LA. Dabrowski K. (1992) Ascorbate concentration in fish ontogeny. Journal of Fish Biology 40, Dhert Ph., Lavens P. & Sorgeloos P. (1992) A simple test Blackwell Science Ltd, Aquaculture Research, 29,

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