INFLUENCE OF β - GLUCAN ON THE GROWTH AND SURVIVAL OF PENAEUS MONODON UNDER DIFFERENT SALINITY CONDITIONS

INFLUENCE OF β - GLUCAN ON THE GROWTH AND SURVIVAL OF PENAEUS MONODON UNDER DIFFERENT SALINITY CONDITIONS Suryakant Patil 1, Ahilan, B 2 * and M.J. Prince Jeyaseelan 3 ABSTRACT The effectiveness β-glucan on the growth and immune response of juveniles of Penaeus monodon (Fabricius) were assessed under three salinity ranges i.e. sea water (30-35ppt); brackishwater (15-20ppt) and near freshwater (<5ppt). The levels of incorporation of β-glucan in the feed were at 0.025%, and. The experimental diets were fed to the juveniles of Penaeus monodon for 60 days. β-glucan incorporated feed (GS2) at level performed excellently and the growth was 1210mg with 75% survival; followed by β-glucan at (GS1 at 0.025%) and the growth was 1005mg with 75% survival. The maximum growth in GS3 at was 991mg with 58.33% survival in sea water. The effect of -glucan on the immune response of Penaeus monodon juveniles to bacterial challenge with Vibrio alginolyticus was studied. It revealed that in the immersion dose of 2.0x105cfu/ ml in the 10 days post challenge study led to better survival when the inclusion level was β-glucan in feed under sea water rearing condition. Key words : Shrimp - immunostimulant- -glucan - Growth - immunity. INTRODUCTION The t iger shri mp, Penaeus monodon accounts for 50% of the shrimp exports from India. Though the shrimp culture sector witnessed a major growth during last decade, production is currently affected seriously by problems linked to environment degradation and dreadful infectious and noninfectious diseases. In shrimps, vibrios are the causative agents for a number of epizootic diseases. Control of disease is a priority, as shrimp production has to be ecologically and economically sustainable. The prophylactic measures to combat shrimp disease outbreaks need an integrated approach. Indiscriminate and frequent use of antibiotics would lead to development of drug resistant strains and in turn could compound the disease problems leading to mortality during out-break (Karunasagar et al., 1994). Moreover, there are possibilities for the transfer of the resistant pathogens associated with farmed shrimps, to human beings (Aoki, 1992). Many developed countries to which India export cultured shrimps have already banned the use of many antibiotics in aquaculture systems. These issues stress the need for counter measures to prevent the out-break of diseases in aquaculture systems, wi thout a negati ve impact o n t he environment. Keeping this in view, the present study was carried out to determine growth performance and disease resistance of Penaeus monodon under different salinity conditions using -glucan. MATERIALS AND METHODS The experiment was designed to study the growth and survival of P.monodon using the immunostimulant -glucan from Saccharomyces cerevisiae at three concentration levels in three 1. Senior Scientist, Central Institute of Fisheries Education, Versova, Mumbai. 2. Professor, Department of Aquaculture, Fisheries College and Research Institute, Thoothukudi. 3. Professor and Head, Department of Aquaculture, Fisheries College and Research Institute, Thoothukudi 198

Suryakant Patil et.al., different salinity regimes i.e. near freshwater (<5ppt), brackishwater (15-20 ppt) and seawater (30-35 ppt). Controls were maintained for the above said salinity regime. The control feed was prepared without incorporating the immunostimulant. The experiment al period was 60 days. The immunostimulant incorporated feed was fed to the shrimps for first 7 days and then these were fed with control feed for 8 days. The above said protocol was maintained up to 60 days. Experimental set up The juveniles of P.monodon were procured from Kanoi hatchery, Trichendur, Tamil Nadu. The seeds were acclimatized and stocked in 300 litres capacity round FRP tank filled with seawater (35ppt). The seeds were acclimati zed for 3 days and subsequently they were acclimatized to <5ppt and 15-20ppt respectively in a span of gradually, 5 days before initiation of the experiment. Statistical analysis Two way analysis of variance (ANOVA) calculation was done to find out variation in the growth related parameters of shrimps treated with different immunomodul ators, as well as the percentage of mortality in immune response study. Growth study RESULT AND DISCUSSION The growth and survival particulars of Penaeus monodon juveniles fed with -glucan are presented in Table 1. An enhanced growth of 1194.57 mg was observed in sea water at the inclusion level of (GS2). Moderate growth as recorded in animals fed with level (GS3) of -glucan incorporated feed (985.43mg) while the group fed with 0.025% (GS1) inclusion level showed 977.69 mg growth. The growth l in the control was 875.68 mg only which was less when compared to the treatments. Preparation of feed A standard shrimp diet was formulated, using the common ingredients and the the dough was pelletized using 1mm diameter die by a hand pelletizer. The control feed was prepared without inclusion of immunostimulant. Pathogenicity studies Patho genic bacterial strains of V.alginolyticus and V.vulnificus isolated from white spo t virus infected shri mp were used in the pathogenicity study. Water quality analysis The physico - chemical parameters of the experimental set up under three salinity regimes were monitored for -glucan experiments following the methodology described in APHA (1976). The growth and survival of Penaeus mondon juveniles (Table 2) in brackishwater (15-20ppt), when fed with -glucan from saccharomyces source, showed an average growth rate of 1115.07mg at (GS2) inclusion level, 990.06mg at (GS3) level and 890.75mg at 0.025% (GS1) level. Immune response in Penaeus monodon juveniles Penaeus monodon juveniles challenged with Vibrio alginolyticus approximating to that of LD50 level (2.0x105cfu/ml) resulted in different levels of mo rt ali ties at different l evels of glucan incorporation (Table 4). In general the shrimps used in controls of different salinities (NFWC, BWC & SWC), when challenged with pathogenic strains of V.alginolyticus and V.vulnificus showed relatively higher mortality in near freshwater condition (<5ppt salinity) and lower mortality in seawater condition. [Exceptionally, the V.vulnificus strain used for -glucan study 199

showed highest mortality of shrimp in control in brackishwater condition]. With reference to -glucan administration to shrimp through feed, it consistantly proved better in term of shrimp growth and immunity enhancement (against secondary vibrio pathogens) In -glucan administration studies on shrimp, with reference to immune response, the experimental group fed with -glucan incorporated diets at and 0.025% levels showed best survival under seawater condition, when challenged with V.alginolyticus. However, with regard to growth studies, best performance in shrimp was achieved only in shrimp under seawater condition and administered with -glucan incorporated feed at level. In the present experiment, the growth rate of Penaeus monodon juveniles fed with -glucan derived from saccharomyces source, in the sea water (30-35ppt) recorded maximum growth at inclusion level of than and control; the survival percentage was also maximum in the inclusion level of 0.1.% (75%). This could be comparable with the study conducted by Rao et al (1994). Pradeepkumar (1996) reported that glucan incorporated feed at level showed maximum growth in P.indicus juveniles when compared to 0.05% and 0.025% levels. Sung et al (1994) recorded increased growth rate of P. monodon post- larvae when su bject ed to immersion in 0.5mg/ml, 1mg/ml and 2mg/ml glucan suspensions for 3 hours. According to Rao et al (1996), when P.monodon juveniles (5-8g) were fed with glucan at different levels (0.2, 0.4, 0.6, and 0.8% level) they showed higher growth rate at 0.4% and 0.6% levels, while in the present study,?-glucan administered different levels (0.025%, and levels) showed relatively higher growth rate of shrimp at inclusion level of only. The positive immunomodulatory effect by glucan in the post-larvae of P.monodon demonstrated by Sung et al (1994), could be comparable to the results obtained in the present study. 200 Influence of - glucan on the growth... In the present investigation?-glucan at level of administration to shrimp in seawater condition showed less mortality (24.99 8.33%) than control (58.33 8.33%) when challenged with pathogenic Vibrio alginolyticus. According to Raju (1998) glucan at relatively higher concentration of 0.4% and 0.7% levels exhibited relatively lower mortality rate (33.33% and 25% respectively) in shrimp juveniles when challenged with Vibrio alginolyticus than control. According to Rao et al (1996), P.monodon juveniles fed with glucan at 0.6% level showed relatively lesser mortality level (66% only). When challenged with Vibrio alginolyticus, corresponding as against mortality level in control. The protective effect of glucan against white spot virus was also reported by Rao et al (1996). Glucan fed animals were exposed to flesh of white spot virus affected shrimps. According to them, the mortality of shrimp was found to be 40% in 0.6% glucan incorporated diet fed experimental group, even after 10 days, while in control, the mortality was found to be 100 percent within 96 hours. It is worthy to mention here that?-glucan administered in the present study provided protect ion agai nst secondary pat ho genic V.alginolyticus strain isolated from white-spot virus infected shrimp. The effect of glucan on the immune response of P.monodon juveniles challenged with a virulent bacterial pathogenic strain of Vibrio vulnificus isolated from white spot virus infected shrimp and with secondary pathogenic strain at immersion dose of 1.75 x 105 cfu/ml resulted in minimum mortality in the present investigation, (ie. 24.99 8.33% at level) in 10 days of post challenge, while in control, the mortality rate of shrimp observed was 83.33%. Sung et al (1994) observed the mean mortality level of shrimp at 20% level at an immersion dose of 0.5mg glucan/ml for 3h, after 18 days of post challenge study with Vibrio vulnificus (probably a normal pathogenic strain) as against 60% in the control.

Suryakant Patil et.al., β-glucan administred through the feed in the present study showed a positive effect on the growth and survival of Penaeus monodon juveniles. Pradeepkumar (1996) reported that the glucan at relatively higher concentration () exhibited lower mortality (33.33%) in P.indicus as against control (66.66%). Raju (1998) observed that the mortality level of P.monodon administered with?- glucan incorporated level (at 0.7% level) was relatively lesser (25.0 0.833%) than the control group (77.77 0.785%) when challenged with pathogenic strain of V.alginolyticus. It is obvious to state that in shrimps, protection against disease provided by glucan is possible due to its activation of the prophenoloxidase (Propo.) system. Song and Sung (1993) observed that M-glucan enhanced the nonspeci fic disease resistance in the P L- 20 of P.monodon, and they observed the protective effect in P.monodon against Vibrio vulnificus in the group treated with 0.5mg and 1mg of M-glucan/ml suspension. But it was not so in the groups treated with 2mg/ml suspension. Itami et al (1994) recorded increased disease resistance in Kuruma shrimp, Penaeus japoni cus agai nst Vi bri os, by oral administration of -1,3-glucan (Schizophyllan, SPG) derived from Schizophyllan commune. According to them, the final survival rate to the level of 90% was observed in 50mg SPG/kg, administered group, 80% in 100mg SPG/kg administered group and 50% of challenged shrimp only in control group. Table - 1 Growth and survival of Penaeus monodon juveniles grown in seawater and fed with β-glucan and control diets β-glucan level Triplicate Initial 15 th day wt (mg) 30 th day 45 th day 60 th day Survival % GS 1 R 1 30.33 92.22 285.36 670.26 952.25 66.66 0.025% R 2 35.25 95.75 290.45 680.85 1005.85 75.00 R 3 38.38 98.36 288.31 689.43 975.45 66.66 GS 2 R 1 40.25 105.65 305.17 701.25 1210.65 75.00 R 2 35.26 110.32 310.25 705.48 1180.82 66.66 R 3 31.33 107.38 315.46 710.32 1192.25 66.66 GS 3 R 1 29.45 80.25 275.75 690.71 991.64 58.33 R 2 25.46 84.75 279.40 685.32 984.32 66.66 R 3 28.36 82.45 272.25 688.50 980.35 66.66 R 1 26.44 70.36 260.75 632.16 880.22 58.33 Control R 2 28.15 75.28 265.40 625.40 875.38 50.00 R 3 30.00 72.35 261.35 628.32 871.45 58.33 201

Table - 2 Growth and survival of Penaeus monodon juveniles grown in brackishwater and fed with β-glucan and control diets β-glucan level GS 1 0.025% Triplicate Influence of - glucan on the growth... Initial 15 th day 30 th day 45 th day 60 th day Survival % R 1 32.35 75.37 265.35 528.36 891.26 75.00 R 2 36.42 80.65 271.48 535.45 895.35 66.66 R 3 30.45 70.35 260.32 525.26 885.64 66.66 GS 2 R 1 38.40 84.48 282.22 575.85 1120.25 75.00 R 2 40.25 86.35 285.87 563.45 1110.85 66.66 R 3 42.33 91.28 290.62 571.36 1114.12 75.00 GS 3 R 1 28.26 82.45 275.36 554.46 1002.38 66.66 R 2 31.44 84.86 270.45 551.86 985.46 75.00 R 3 32.45 78.79 268.19 546.42 982.36 66.66 R 1 28.26 65.45 250.35 521.25 830.84 50.00 Control R 2 30.68 60.38 254.86 525.75 825.58 58.33 R 3 32.25 68.86 261.35 529.26 835.75 58.33 Table - 3 Growth and survival of Penaeus monodon juveniles grown in near freshwater condition (<5ppt) and fed with β-glucan and control diets β-glucan level Triplicate Initial 15 th day 30 th day 45 th day 60 th day Survival % GS 1 0.025% R 1 24.16 70.28 161.32 335.32 740.25 75.00 R 2 35.25 79.25 165.38 340.48 735.38 66.00 R 3 33.33 72.35 154.50 342.85 748.85 58.33 GS 2 R 1 38.45 98.86 190.46 365.24 825.52 66.66 R 2 35.86 95.45 184.35 360.82 838.26 75.00 R 3 32.33 91.36 180.82 356.45 816.85 66.66 GS 3 R 1 36.25 65.45 175.45 350.36 685.38 58.33 R 2 34.45 60.38 170.36 347.95 681.56 66.66 R 3 32.38 63.35 167.45 345.28 682.35 66.66 Control R 1 28.32 54.40 150.38 325.45 650.28 58.33 R 2 24.45 50.86 146.35 321.38 654.86 50.00 R 3 22.86 48.40 142.85 316.25 658.25 50.00 202

Suryakant Patil et.al., Table - 4 Effect of β-glucan on the immune response of Penaeus monodon juveniles to bacterial challenge with Vibrio alginolyticus (Immersion dose: 2.00 x 105 cfu/ml) under different salinity conditions Sl. Number dead /Number challenged Percentage mortality Treatment No Trial I Trial II (Mean ± SD) 1. SWGS 1 0.025 % 1/6 2/6 24.99 ± 8.33 2. BWGS 1 0.025 % 2/6 2/6 33.33 ± 0.00 3. NFWGS 1 0.025 % 3/6 2/6 41.66 ± 8.33 4. SWGS 2 2/6 1/6 24.99 ± 8.33 5. BWGS 2 2/6 2/6 33.33 ± 0.00 6. NFWGS 2 3/6 2/6 41.66 ± 8.33 7. SWGS 3 2/6 2/6 33.33 ± 0.00 8. BWGS 3 3/6 3/6 50.00 ± 0.00 9. NFWGS 3 2/6 3/6 41.66 ± 8.33 10. SWC 3/6 4/6 58.33 ± 8.33 11. BWC 4/6 4/6 66.66 ± 0.00 12. NFWC 5/6 4/6 74.99 ± 8.33 \In 10 days post challenge SW -Seawater BW - Brackishwater NFW -Near freshwater condition (<5ppt) REFERENCES M.F.Sc., Thesis, Tamil Nadu Veterinary and Animal Sciences University. pp.69 (M.J. Prince Aoki, T., 1992. Chemotherapy and drug resistance Jeyaseelan). in fi sh farms in Japan. In: Shari ff, M., Subasinghe, R.P., Arthur, J.R. (eds) Disease in Raju.V.S.R.K., 1998. Effect of select immunostimulants on the growth and immune response of Penaeus Asian Aquaculture 1, Asian Fisheries Society, monodon (Fabricius), M.F.Sc., Thesis, Tamil Manila, Philippines, pp. 531-546. Nadu Veteri nary and Animal Sciences APHA., 1976. Standard methods for the examination University. pp67 (M.J. Prince Jeyaseelan). of water and wastewater, American Public Rao, A.V.P., D.Panchayuthapani, M.Anitha and B.S. Health Association. Ajithakumar, 1996. Resistance to diseases in Karunasagar, I., R.Pai, G.R. Malathi and I. Karunasagar, tiger shrimp, Penaeus monodon t hrough 1994. Mass mortality of Penaeus monodon larvae incorporation of glucan in feed. Fishing due to antibiotic-resistant Vibrio harveyi Chimes, 16 : 41-42. infection. Aquaculture, 128: 203-209. Sung, H.H., G.H. Kou and Y.L. Song, 1994. Vibriosis Pradeepkumar P., 1996. The effect of feeding resistance induced by glucan treatment in tiger selected immunomodulators on the growth and shrimp (Penaeus monodon). Fish Pathology, immune response in penaeid shrimp juveniles, 29(1): 11-17. 203