, ±0.36 g ACP AKP SOD UL WSSV S963.73

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1 1,2 1 1,3 1,4 1., , , %1%0.1%, 34.83±0.36g d(PO) ACPAKPSODUL 22d0.0042kg/kgWhite Spot Syndrome Virus, WSSV POACPAKP ULSODWSSV 0.1%WSSV14d 63.3%±5.8%96.7%±7.3%, WSSV S Effect of photobacterium Damselae subsp. damselae on the non-specific immune response and disease resistance of Litopenaeus vannamei LAN Ping 1,2 SONG Xiao-ling 1 ZHANG Hui 1,3 CHENG Jun-jun 1,4 HUANG Jie 1 ( 1 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao ) ( 2 Shanghai Ocean University, ) ( 3 Dalian Fisheries University, ) ( 4 Wuhan Institute of Virology, Chinese Academy of Sciences, ) ABSTRACT Litopenaeus vannamei at 4.83±0.36g body weight were fed with 0.1% heat-killed, 1% heat-killed and 0.1% living photobacterium Damselae subsp. damselae for 21 days respectively. The serum was collected at 0, 5, 10, 15 and 20 days post-immunization. Phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) and lysozyme activity (UL) in the serum of L. vannamei were determined to evaluate the non-specific immune effect of D. subsp. damselae. After 21 days, the shrimps were challenged by white spot syndrome virus (WSSV) and the mortalities of challenging test were recorded. The results showed that most PO, ACP, AKP, UL and SOD in the serum of L. vannamei of the test groups were significantly different (P<0.05 or P<0.01), and the resistance ability of shrimps to WSSV was also significantly enhanced by the diet supplemented with D. subsp. damsela. The group fed with 10g heat-killed D. subsp. damsela per 1kg shrimps exhibited the strongest resistance to WSSV in 14 days and its final cumulative mortality was 63.3±5.8%, while the control was 96.7±7.3%. The results indicated that D. subsp. damselae improved shrimp non-specific immunity and its resistance to WSSV, suggesting that it might be a promising AA CB lanping0524@163.com,Tel: songxl@ysfri.ac.cn

2 diet supplement in shrimp farming. KEY WORDS Litopenaeus vanname Photobacterium Damselae subsp. Damselae Non-special immunity Resistance to disease White Spot Syndrome Virus (WSSV) ( 2007), (Lopez et al. 2003Montero-Rocha et al ),, 2006 ), 2008 ) 20 5POACP AKPSODULWSSV WSSV WSSV (Litopenaeus vannamei) ±1.18cm,4.83±0.36g L1 1/ /5001h 22~25pH8.2±0.2DO 7.0 mg/ml7d (Photobacterium damselae subsp. damselae) COD 600nm CFU/ml, 160,, 4010% A BC 0.1%1%D0.1% Table 1 Composition of the basic diet

3 Component (g/kg) Content (g/kg) Vitamin mixture 10 C Vitamin C 2 NaH 2 PO 4 2H 2 O 2 Na 2 HPO 4 12H 2 O 3 Sodium alginate 10 Vegetable oil 20 Peanuts powder 250 Soybean powder 250 Shrimp powder 100 Flour 30 Corn powder 30 Fishmeal d,10-151mL 1.5mL PO 2001Ashida (Unit/mg Protein)PO=10 3 AA/CC UL Micrococcus lysoleikticus 0.1molpH=6.4OD ~0.5190µL 10µL570nmA37 C30min 10minAUL=AA/A ACP (U/100mL)=A /A AKP U/100mL =A/A SOD,SOD SOD50%SOD1USODU/mL =AA/A 50% 1.4 WSSV WSSV-80 C WSSV L

4 -80 C 14d 1.5 SPSS13.0Duncan PO 120PO 53PO(P<0.05) ,BCPOD15PO 2020PO 5BDPO POU.mg-1) PO activity /d 1 Fig. 1 Serum phenoloxidase activity of L. vannamei in the test groups :, P<0.05, AB0.1%C 1%D0.1% same letter means no significant difference between the groups. addition of 0.1% living D. subsp. damselae 2.2 SOD 253SODP< SODP<0.0515D SOD3P<

5 SOD % SOD SODU.ml-1) SOD activity /d Fig. 2 2 Serum superoxide dismutase activity of L. vannamei in the test groups :, P<0.05, A, B0.1%C 1%D0.1% same letter means no significant difference between these four groups.. addition of 0.1% living D. subsp. damselae 2.3 UL 3BC20UL P>0.05D20 P< %UL U.ml -1 ) UL activity /d 3

6 Fig. 3 Serum lysozyme activity of L. vannamei in test groups :, P<0.05, A, B0.1%C 1%D0.1% same letter means no significant difference between these four groups.. addition of 0.1% living D. subsp. damselae 2.4 ACP 4,53ACP P<0.0510DACPACP 3P< ACP DP<0.05P<0.05 DACP1520B,CP< % P<0.05 ACP/ ACP activity /d 4 Fig. 4 Serum acidic phosphatase activity of L. vannamei in test groups :,P<0.05, A,B0.1%C 1%D0.1% same letter means no significant difference between these four groups.. addition of 0.1% living D. subsp. Damselae 2.5 AKP 5BCAKP5 15D P<0.05,10,0.1% AKPBCP<0.05

7 AKP/ AKP activity /d Fig. 5 5 Serum alkaline phosphatase activity of L. vannamei in test groups :,P<0.05,A, B0.1% C 1%D0.1% same letter means no significant difference between these four groups. addition of 0.1% living D. subsp. Damselae 2.6 WSSV 66 66~10A BDP<0.0510A 96.7%±7.3%B76.7%±8.1%D(63.3%±5.8%)P<0.05C 88.3%±6.9%ABDP>0.05, 0.1%WSSV % Cumulative Mortalit A B C D d Infection 6 WSSV Fig. 6 Cumulative mortality of L. vannamei after challenged with WSSV :A,B0.1%C 1%D 0.1%

8 Note: addition of 0.1% living D. subsp. damselae 3 3.1, 2008 Rengpipat1998Bacillus S11() Thyssen et al.2000; Osorio et al. 2005; Kijima et al ,, 204 3, 0.1% 3.2 Van Hulten et al ~10d WSSV WSSVCBD20 POU L, CB %

9 Chang2003-1,3-glucan20WSSV 80% WSSV6 63.3%Chang, 2005WSSV WSSV 4 WSSV 0.1%,,,,, ,272: ,,, , 4:55-58.,,,,, ,275:51-55.,,,,.2008.Lactobacillus spplh., 2711: ,[],,,2008,,, ,292:48-52.,,,,.2007.A3-α-.,281:59-64.,,,, A3-α-.,158: ,,,,, ,15 2: ,,,.2001.(WSSV).,84: Chang C F,Su M S,Chen H Y Dietary ß-1,3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus.fish and Shellfish Immunol,15: Kijima M T,Kowanishi M,Fukuda Y.2007.Molecular diversity of Photobacterium damselae sp.piscicida from cultured amberjacks Seriola spp.in Japan by pulsed.field gel electrophoresis and plasmid profiles.journal of applied microbiology,103: Lopez A,Pierce,G J,Santos M B.2003.Fishery by-catches of marine mammals in Galician waters results from on-board observations and an interview survey of fishermen.biological Conservation, 111: Montero-Rocha A D,McIntosh R, Sanchez-Merino.Immunostimulation of white shrimp (Litopenaeus vannamei) following dietary administration of Ergosan. Invertebrate Pathol,2006,91: Osorio,C R,Collins,M D,Romalde,J L.2005.Variation in 16 S - 23 S rrna Intergenic Spacer Regions in Photobacterium damselae:a Mosaic-like Structure.Applied and Environmental Microbiology,712: Rengpipat S,Phianphak W,Piyatiratitivorakul S.1998.Effects of a probiotic bacterium on black tiger shrimp Penaeus monodon survival and growth.aquaculture,167 (3-4) : Thyssen,A.,van Eygen,S,Hauben L.2000.Application of AFLP for taxonomic and epidemiological studies of Photobacterium damselae subsp.piscicida.international Journal of Systematic and Evolutionary Microbiology,50: Van Hulten M C,Westenberg M,Goodall S D.2000.Identification of two major virion protein genes of white spots syndrome virus of shrimp.virology,266 2:227236

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