Effects of Astaxanthin on Larval Growth and Survival of the Giant Tiger Prawn, Penaeus monodon

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1 Effects of Astaxanthin on Larval Growth and Survival of the Giant Tiger Prawn, Penaeus monodon Jintana Darachai, * Somkiat Piyatiratitivorakul, Prasat Kittakoop Charoen Nitithamyong, Piamsak Menasveta Aquatic Resources Research Institute, Department of Marine Science and Marine Biotechnology Research Unit, Chulalongkorn University, Bangkok 10330, Thailand ABSTRACT: The aim of this study was to evaluate effects of natural and synthetic astaxanthins on growth, survival and low salinity resistance of Penaeus monodon larvae. Four diets: algal astaxanthin-added diet (AAD), synthetic astaxanthin-added diet (SAD), non astaxanthin supplemented diet (NAD) and natural food (NF) were prepared to feed three P. monodon larval stages (zoea, mysis and postlarvae). The results indicated that zoea fed AAD, NF and NAD survived significantly better than zoea fed SAD. For the mysis stage, larvae fed NF and AAD had better survival rates than those fed NAD and SAD. After 15 days of rearing the postlarval stage to PL- 15, AAD showed the best survival rate and it was significantly higher than that of PL-15 that had been fed NF. PL-15 fed AAD and NF were significantly longer than those fed SAD and NAD (P<0.05). In low salinity challenge tests, the postlarvae fed AAD showed higher tolerance than postlarvae fed other diets. KEY WORDS: astaxanthin, Penaeus monodon, larvae, growth, survival, Haematococcus pluvialis INTRODUCTION Larviculture of marine shrimp has become increasingly important due to an expansion of shrimp culture in coastal zones. Each year in Thailand, at least 40 billion postlarvae of Penaeus monodon are needed to support shrimp aquaculture. In order to ensure good quality shrimp postlarvae, good quality food, either natural or artificial needs to be used. Since it is difficult or impossible to control natural live feeds which vary in quality seasonally, larval feed developments have focused on artificial feeds. These artificial feeds are still in need of more intensive study. One of the components of artificial feeds is astaxanthin, the main carotenoid found in the integument of shrimp. It is an orange to red carotenoid pigment that contributes to consumer appeal for foods in the marketplace. However, it is also important for health, since it is involved in intra-cellular protection by stabilizing cell membranes and improving health and immunology through the removal of oxygen free radicals (Roche News 1993). Since animals lack the ability to synthesize carotenoids, the pigments must be included in thier feed. There is considerable interest in using natural sources of astaxanthin for feed, because synthetic astaxanthin is expensive and difficult to prepare. Algae in the genus Haematococcus are known to produce astaxanthin. H. pluvialis has been especially studied because of its rapid growth and proficient production of astaxanthin (Johnson & An 1991). It can be used to produce the carotenoid in large quantity, free from toxicity. In the present experiments, natural astaxanthin from H. pluvialis was produced and compared with synthetic astaxanthin for effects on shrimp larval health, growth and survival. MATERIALS AND METHODS Algal culture for astaxanthin H. pluvialis NIES144 obtained from the National Institute for Environmental Studies (NIES), Japan was cultured under optimal conditions for growth (temperature 25 o C, 10 h in darkness and 14 h at 1.5 klux illumination). When the culture reached 10 6 cells per ml, the cells were continuously stressed with 10 klux illumination to stimulate astaxanthin accumulation. Effect of astaxanthin on P. monodon larvae The present study was carried out at the Department of Marine Science and the Marine Biotechnology Research Unit, Chulalongkorn University. A completely randomized design was used for four diets with five replicates. The diets were; natural food (NF: Chaetoceros sp. for zoea and the first mysis stages, and newly hatched Artemia salina for mysis II to postlarva-15), control diet (NAD: a semi-purified diet that was astaxanthin-free), algal astaxanthin-added diet (AAD: a semi-purified diet with added astaxanthin from H. pluvialis) and a synthetic astaxanthin-added diet (SAD: a semi-purified diet with added synthetic astaxanthin). The amount of astaxanthin added in the diet was controlled at 200 mg/kg diet. The feeding experiments were carried out at three larval stages; zoea I-III, mysis I-III and postlarvae P. monodon nauplii obtained from a commercial hatchery in Chonburi Province were used in this experiment. Nauplii parents were from Andaman Sea stock. The feeding tests were started at the beginning of zoea I, mysis I and Darachai J, Piyatiratitivorakul S, Kittakoop P, Nitithamyong C, Menasveta P (1998) Effects of astaxanthin on larval growth and survival of the giant tiger prawn, Penaeus monodon. In Flegel TW (ed) Advances in shrimp biotechnology. National Center for Genetic Engineering and Biotechnology, Bangkok. * psomkiat@chula.ac.th

2 118 Darachai et al. postlarva-1, respectively. The initial larval density for each stage was 100 larvae/l for zoea, 80 larvae/l for mysis and 30 larvae/l for postlarvae. The larval rearing units consisted of conical shaped aquaria containing 4 l sea water. The rearing system was static with partial water change (about 2/3 of total volume) every morning. Filtered and chlorinated seawater at 30 ppt salinity was used and water temperature was o C. All rearing units were provided with gentle, continuous aeration. All larval stages were fed ad libitum with five feed additions; 08:00, 11:00, 14:00, 17:00 and 20:00 h. The first feed for zoea I in all treatment groups was Chaetoceros sp. and then the designed diets were substituted as appropriate for the later developmental stages. Sizes of the semi-purified diet particles used in the experiments were < 106 microns for zoea and mysis and microns for postlarvae. Table 1. Percentage composition of the experimental diets. Ingredients % Use NAD AAD SAD Casein Wheat flour Fish oil (refined tuna) Na- citrate Na-succinate Glucosamine-HCl Mineral mix Vitamin mix* Cholesterol 90%* Lecithin Rice bran Carrageenan Synthetic astaxanthin* Cysts of H. pluvialis Wheat flour** *Provided by Rovithai Ltd., Thailand.; **For protein compensation; Mineral mix 100 g contains; K 2 HPO g, CaSO g, MgSO 4.7H 2 O 3.04 g and Na 2 HPO 4.2H g; NAC = no added astaxanthin control diet; AAD = algal astaxanthin diet; SAD = synthetic astaxanthin diet Experimental diets The basal diet in these experiments was in a semi-purified, microparticulate form. The diet was modified from Teshima and Kanazawa (1984) and the composition is shown in Table 1. Astaxanthin sources were Carophyll Pink (synthetic carotenoid, 8% astaxanthin, coated with gelatin, carbohydrates and antioxidants and provided by Roche Ltd., Switzerland) and algal astaxanthin (astaxanthin produced by H. pluvialis NIES144, 1.2% astaxanthin with 38% protein). The control diet contained no supplemental carotenoid. The amount of wheat flour was altered to compensate for astaxanthin elimination and for added protein from the algae. The natural diet used consisted of Chaetoceros sp. and newly hatched Artemia sp. Salinity stress tests Postlarvae-15 fed different diets were challenged in stress tests. Ten larvae from each treatment replication were immediately transferred from rearing salinity (30 ppt) to 2 ppt salinity. Then, the number of dead shrimp was recorded at 10 min intervals for a period of two hours. Data collection Survival of the larvae for each dietary treatment was recorded when the larvae reached the early phase of the next developmental stage. Survival rate was calculated as the final number surviving as a proportion of the initial number of larvae in the test. For effect of diets on growth, ten postlarvae- 15 from each replicate were randomly sampled for length measurement. The length was determined from the tip of telson to rostrum under a x10 magnification projection microscope. Water quality such as salinity and temperature was determined daily, while ammonium, nitrate and ph were monitored every 2 days. The astaxanthin content in H. pluvialis cysts, in synthetic astaxanthin, in diets and in shrimp tissues was determined using the spectrophotometric technique described by Boussiba and Vonshak (1991) and Davies (1976) and by HPLC (Weber 1990). Statistical analysis All experimental data were analyzed using the Statistic Analysis System (SAS) Program (SAS 1985) or the SPSS- PC program for probit analysis. Means were compared using Duncan s new multiple-range test. RESULTS Cultivation of H. pluvialis NIES144 During cultivation for growth, the H. pluvialis cells were green, biflagellates swimmers with a size of approximately 10µ. The specific growth rate (µ) and doubling time were 0.26 and 2.67 days, respectively. After culturing up to 25 days, the density of the cells reached cells/ml. Encystment was then promoted by increasing the light intensity to 10 klux. The cells lost their flagellae and formed persistent thick walled cysts. Simultaneously, they accumulated massive amounts of astaxanthin. Astaxanthin deposition first occurred around the nucleus and then proceeded radially until the entire protoplast turned red. The size of the cysts was 50 µ (4-5 times larger than the vegetative cells). Fully matured cysts only were harvested days after initiating cell stress. The yield of dried cysts was approximate 0.2 g/l. Freeze dried cyst composition was protein 39.14%, fat 1%, carbohydrate 17.01%, ash 3.25% and moisture 7.45%. The average astaxanthin quantity produced of H. pluvialis was % of algal dry weight. Proximate analysis of diets Proximate analysis of the artificial diets gave protein 47%, fat 8%, fiber 1%, ash 7% and moisture 5%. The astaxanthin content in the algal astaxanthin-added diet (AAD), the synthetic astaxanthin-added diet (SAD), and the control diet (NAD) was , and 0 ppm, respectively. Effect of astaxanthin on survival Survival of shrimp zoea, mysis and postlarvae fed different diets are shown in Table 2. Survivals of zoea fed NF, NAD, and AAD were significantly higher than survival of zoea fed SAD (P<0.05). At the mysis stage, the survival of larvae fed AAD and NF was similar but significantly higher than that of mysis fed NAD and SAD. At the postlarval stage, the best survival was obtained with AAD and this was sig-

3 Effects of astaxanthin on larval growth and survival nificantly higher than that of postlarvae fed NF. However, there were no significant differences in survival amongst larval groups fed AAD, SAD and NAD. Table 2. Percent survival of Penaeus monodon larval stages fed different diets. Survival of larval stage (%) Diets Zoea Mysis Postlarvae NF * 82.0 ± 3.30 a 76.7 ± 8.61 a 55.2 ± 6.14 b NAD 74.0 ± 5.19 a 57.3 ± 5.01 b 68.6 ± 5.73 ab AAD 82.5 ± 3.53 a 69.7 ± a 76.0 ± 9.46 a SAD 27.8 ± 4.01 b 58.1 ± 0.29 b 64.4 ± ab Means in the same column with different superscripts are significantly different (P<0.05); *NF (Natural food) from zoea I to mysis II was Chaetoceros sp. and for mysis II to postlarva-15 was newly hatched Artemia salina. Effect of astaxanthin on growth Growth as measured in terms of total length of postlarva 15 fed different sources of astaxanthin diet are shown in Table 3. The mean lengths for larval groups fed NAD and SAD were significantly lower than those for larvae fed AAD and NF (P<0.05) but there was no significant difference between the groups fed NF and AAD. Table 3. Length of Penaeus monodon postlarva-15 fed different diets % CM for shrimp fed AAD was longer than that for shrimp fed SAD, NAD and NF. Table 4. Time to 50% cumulative mortality (CM) in low salinity resistance test of larvae fed different diets. Diets Time to 50% CM (min) NF NAD AAD SAD Water quality The water quality of larval rearing units for all treatment groups is shown in Table 5. Water quality parameters were similar for the four dietary treatments and were in the normal range for shrimp cultivation throughout the whole period of the experiment. Carotenoids in postlarvae fed different diets Because of their small size, carotenoid analysis of the postlarva 15 was determined on a wet weight basis using a spectrophotometric method. The results of the carotenoid assay are shown in Table 6. Carotenoids in all the diet groups were significantly different (P<0.05). The highest carotenoid accumulation occurred in the shrimp fed NF, followed by the shrimp fed AAD, SAD and NAD, in order of highest to lowest accumulation. Diets Length of PL 15 (cm) Mean ± SD NF ± ab NAD ± b AAD ± a SAD ± b Means with different superscripts are significantly different at P<0.05 Low salinity tolerance Larvae from all of the diet treatment groups started to die within 10 minutes of beginning the test. In the first hour, the highest cumulative mortality (CM) was for the larval group fed NF (76%), followed by those fed NAD (68%), SAD (66%) and AAD (62%). The mortality rate declined after the first hour. At 90 minutes, CM for larvae fed AAD and SAD was 76%, while it was 80% and 86%, respectively for those fed NF and NAD. After 2 h, remaining larvae from all the treatment groups had adapted to 2 ppt salinity and showed only slight mortality up to 140 minutes. Using probit analysis, the effect of diet on time to 50% CM for postlarva 15 was determined and the results are shown in Table 4. They indicated that time to Table 6. Carotenoid concentration (µg/g body wet weight) in PL-15 of Penaeus monodon fed different diets. Source feed Shrimp carotenoid content (ppm) NF ± 0.65 a NAD ± 3.42 b AAD ± 5.62 c SAD ± 0.47 d Means with different superscripts are significantly different at P<0.05 Table 5. Water quality of the rearing units for different stages of Penaeus monodon larvae. Diets Larval stages Ammonium (mg/l) Nitrate (mg/l) ph Temp. ( o C) Salinity (ppt) Natural Z 1 -Z diet M 1- M PL 1 -PL Artificial Z 1 -Z diets M 1- M PL 1 -PL

4 120 Darachai et al. DISCUSSION Haematococcus cultivation H. pluvialis grew well under laboratory conditions at 25 o C. With high light intensity stress, it accumulated astaxanthin and turned red. Astaxanthin esters are the primary pigment in H. pluvialis cysts, typically ranging from 60% to 80% by weight of the total pigment content (Spencer 1989). Kobayashi et al. (1992) showed that H. pluvialis NIES144 accumulated astaxanthin mainly in esterified form [monoester (69%) and diester (20%)]. By contrast, free astaxanthin is the major compound in synthetic astaxanthin. The predominant configuration isomer in H. pluvialis is 3S, 3 S (Johnson & An 1991). This 3S, 3 S configuration is also found in lobster (Homarus gammarus) eggs and yeast (Phaffia rhodozyma). Effect of dietary astaxanthin on shrimp larvae From our results, the highest survival rate of zoea and mysis was obtained with shrimp fed AAD followed by NF, NAD and SAD, in descending order. This indicated that shrimp larvae accept natural better than synthetic astaxanthin. Commonly, the early stages of shrimp larvae are fed with phytoplankton (e.g., Chaetoceros sp.) and zooplankton (e.g., Artemia sp.) from which they derive natural carotenoids (and convert them to astaxanthin). In the case of AAD, it contained H. pluvialis that had produced natural astaxanthin and accumulated various nutrients (from the rich cultivation medium) and these could be utilized directly by the larvae. By contrast, the free form of astaxanthin in SAD may have been difficult for early larval stages to utilize and this may have affected their survival. Indeed, the zoea fed SAD had the lowest survival. In postlarval stages, there were no significant differences in survival rate for larval groups fed AAD, SAD and NAD. The postlarvae fed NF had the lowest survival (significantly lower than the postlarvae on the AAD diet). This indicated that Artemia nauplii as the sole feed to postlarvae could not provide sufficient nutrients for good survival. Chindamaikul and Phimonchinda (1990) reported that the survival of P. monodon from zoea to postlarva-15 was higher when they were fed natural feed (Chaetoceros sp. and Artemia sp.) plus artificial feed than it was when they were fed only natural feed or only artificial feed. Efficacy of astaxanthin was higher for postlarvae fed AAD than for those fed NF (Artrmia sp.). Tanaka et al. (1975) explained that Artemia sp. accumulated mainly canthaxanthin and that two biochemical steps were required for shrimp to convert it to astaxanthin. By contrast, the astaxanthin in H. pluvialis cysts could be used directly. The source of astaxanthin may affect survival rate of shrimp larvae differently at different stages. For example, the survival of early larval stages fed the diet containing synthetic astaxanthin (SAD) was poor but survival was higher for larvae at the postlarval stage. Utilization of free astaxanthin may be poor for zoea and mysis but may be better for postlarvae. Both zoeal and mysis stages naturally consume algae and small living organisms (e.g., rotifers and Artemia) which are natural sources of astaxanthin. The postlarva-15 fed natural diets containing natural astaxanthin were larger than those fed diets containing synthetic astaxanthin or no astaxanthin. The best postlarval growth was in the group fed AAD and this was significantly better than that for groups fed SAD. This indicated that astaxanthin from H. pluvialis (mostly in esterified form) performed significantly better than free, synthetic astaxanthin. Brown et al. (1991) reported that shrimp fed a dry diet plus algae grew faster and had better survival than shrimp fed a dry diet only. This was probably because the algae contained essential nutrients that were lacking in the dry diet. Similarly, H. pluvialis NIES144 was cultured in a rich medium containing thiamin as a growth factor and its dry cysts contained approximately 40% protein and 1% fat. These factors may explain why H. pluvialis cysts in the diet were more advantageous than synthetic pigments. Cohen (1986) reported that dietary xanthophylls from algae are incorporated into the exoskeleton of prawns and lobsters (as astaxanthin) where they play a major role in pigmentation. Other functions are poorly defined. Boonyaratpalin et al. (1994) found that addition of synthetic astaxanthin and canthaxanthin pigment to diets did not effect growth and feed efficiency of P. monodon juveniles. The pigment content of shrimp fed pigment-free diets was less than that of groups fed pigmented diets. This confirmed that shrimp larvae can accumulate carotenoids (mainly astaxanthin) from their diets. The astaxanthin content of shrimp fed NF (Artemia sp.) was higher than that of other groups. It may because Artemia sp. contains various carotenoids, such as astaxanthin, violaxanthin, zeaxanthin, echinenone, b-carotene, lutein in addition to its main pigment canthaxanthin. Some of these carotenoids can be converted to astaxanthin. Moreover, all carotenoids have similar maximum absorption wavelengths that may overlap or interfere during analysis by spectrophotometer. The larvae fed AAD contained higher amounts of carotenoids. This may have resulted for several reasons. For example, shrimp larvae may gain esterified astaxanthin and accumulate it in body tissue better than free astaxanthin. Secondly, the larvae may be more accustomed to natural astaxanthin than synthetic astaxanthin. Third, they may have accept AAD better than SAD. However, the factors controlling pigment absorption, transportation and excretion among various shrimp tissues are not known. Herring (1969) speculated that pigment absorbed in excess an animal s requirement is later excreted. Determination of 50% cumulative mortality upon low salinity challenge showed that larvae fed AAD endured better than larvae fed NF, SAD and NAD. In addition, tolerance of shrimp fed pigmented diets (AAD and SAD) was higher than those fed a pigment-free diet (NAD). Astaxanthin seemed to be helpful to the postlarvae and to prolong their life upon this acute environmental stress. The esterified astaxanthin in AAD was a storage form of the pigment and it was probably accumulated in the lipid portion of the larval hepatopancreas before transfer to other sites in the body by haemolymph (Ghidalia 1985). This was probably also true for the free astaxanthin in SAD. However, P. monodon has been reported to accumulate esterified astaxanthin (85.7%) more than free astaxanthin (14.3%) (Latscha 1989).

5 Effects of astaxanthin on larval growth and survival Because astaxanthins contain a long conjugated double bond system, they are relatively unstable and usually scavange oxygen radicles in cells (Stanier et al. 1971). When the shrimp were transferred from high to low salinity, they were exposed to stress and needed to expend more energy to maintain osmotic stability. There would have been the potential for generation abnormally high levels of oxygen radicles. However, the dynamics of this interaction and how it relates to adequate or excess astaxanthin content in a cell were not examined in this study. Although the mechanism by which astaxanthin improved the response to stress cannot be explained, the information that natural astaxanthin (from H. pluvialis) is more efficacious than synthetic astaxanthin for growth, survival and stress resistance of shrimp larvae should be useful for further research on shrimp larval nutrition. Acknowledgements. This research was supported by a grant from the Thai National Center for Genetic Engineering and Biotechnology. The authors would like to thank Mr. Sari Donnau for his technical support. LITERATURE CITED Boonyaratpalin M, Wannagowat J, Borisut C, Boonchuay S (1994) Effect of various dietary canthaxanthin and astaxanthin levels on pigmentation of giant tiger shrimp. Technical paper no.18/ National Institute of Coastal Aquaculture, Songkhla (in Thai). Boussiba S, Vonshak A (1991) Astaxanthin accumulation in the green alga Haematococcus pluvialis. Plant Cell Physiol. 32(7) : Brown MR, Jeffrey SW, Garland CD (1991) Nutritional aspects of microalgae used in mariculture; A literature review. Fishing Industry Research and Development, Trust Account 86/81. Department of Primary Industry, Canberra, ACT. Chindamaikul T, Phimonchinda T (1990) The studies on comparison of nursing Penaeus monodon Fabricius with different feed. Technical paper No. 34/1990, Phuket Coastal Aquaculture, Department of Fisheries (in Thai). 121 Cohen Z (1986) Products from microalgae. Handbook of Microalgal Mass Culture. Richmond ed. Florida: CRC Press, pp Davies BH (1976) Carotenoids. Chemistry and Biochemistry of Plants Pigments, 2 nd ed, vol 2. London: Academic Press Ghidalia W (1985) Structural and Biological Aspects of Pigment. In: Bliss DE, Mentel LH (eds). The Biology of Crustacea. vol 9, Integument, Pigment and Hormonal Processes. London: Academic Press Herring P (1969) Pigmentation and carotenoid metabolism of the marine isopod Idotea metallica. Mar. Bio. Ass. UK. 49: Johnson EA, An GH (1991) Astaxanthin from microbial sources. Critical Reviews in Biotechnology 11(4): Kobayashi M, Kakizono T, Yamagushi K, Nishiro N, Nagai N (1992) Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions. Fermentation and Bioengineering 74(1): Latscha T (1989) The role of astaxanthin in shrimp pigmentation. Advances in Tropical Aquaculture Roche News (1993) Roche Aquaculture Center, Vol. 1/1993. Bangkok. SAS, The Statistic Analysis System, SAS Institute Inc. USA, 95 pp Spencer K (1989) Pigmentation supplements for animal feed composition. US Patent No. 4,871,551 Stanier RY, Kunizawa MM, Cohen-Bazire G (1971) Purification and property of unicellular blue-green algae (Order Chroococales). Bacterial Rev. 35: Tanaka Y, Malsuguchi H, Katayama T, Simpson KL, Chichester CO (1975) The biosynthesis of astaxanthin-xviii. The metabolism of the carotenoids in the prawn Penaeus japonicus Bate. Bull. Jpn. Soc. Sci. Fish. 42 (2): Teshima S, Kanazawa A (1984) Effects of protein, lipid and carbohydrate levels in purified diets on growth and survival rates of the prawn larvae. Bull. Jpn Soc. Sci. Fish. 50 (10): Weber S (1990) Determination of added stabilized astaxanthin in fish feeds and premix with HPLC. Roche-publication, Index No. 2101, 59-61