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1 2015 7, 22(4): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 庚宸帆, 田燚, 张宇鹏, 常亚青, 宋坚,, 摘要 :, 2 (Apostichopus japonicus), (16.93±3.08) g, 30 6 h 3 18, h, 30, 24 h, Na + K + Cl, Na + K + Cl 18, (131.15±14.42) mmol/l, (6.08±0.24) mmol/l (141.76±2.13) mmol/l; Ca 2+ Na + -K + -ATP 18 4 d (P<0.05), Na + -K + -ATP,, Na + K + Cl, : ; ; ; Na + -K + -ATP ; 中图分类号 : S917 文献标志码 : A 文章编号 : (2015) , (Rachycentron canadum Linnaeus) Na + -K + -ATP [1] (Epinephelus coioides), Na + -K + -ATP [2] (Acipenser schrenckii), Na + Cl, [3] (Oncorhynchus keta),, [4] (Apostichpus japonicus),, [5], [6] [7] [8] (SOD) (LYZ) (ACP) [9] [10] [11],, [12], 收稿日期 : ; 修订日期 : 基金项目 : ( ); 863 (2012AA10A412); (LJQ ). 作者简介 : (1990 ),,. gengchenfan@163.com 通信作者 : 燚,,,,. tianyi@dlou.edu.cn

2 4 : 667, [13] [14 16] [17], [18],,,,, 18, 6 h 3 (, 30D 24D 18D, h ); h ( 18U); ( ) 18 30, 6 h 3 ( 24U 30U, ), 3 1, 3, 1 ml,, 1.5 ml, , (16.93±3.08) g,, : Gonotec Osmomat 030, Olympic, Epoch biotech, 96 (JET BIOFIL) 96well, Hitachi,,, YSI, L, 24 h : 23.8~24.1, ph 7.9~8.3,, 3.6 mg/l 1, 5% 1.2.2, 3 : ( ): (31.09±0.12) 1 Fig. 1 Diagram of the salinity change patterns , 3 (1) :, Na nm OD : [Na + 测定孔 OD值空白孔 OD 值 ](mmol/l)= 标准孔 OD值空白孔 OD 值 (140 mmol/l) (2) :, K +

3 (NA-TPB) 450 nm OD, 0.4 mmol/l (3) : Ca 2+ (MTB), ; 610 nm, 1 mmol/l (4) Cl, Cl, 480 nm, OD, 20 mmol/l (5) : 测定孔 OD值空白孔 OD值 (g/l)= 标准孔 OD值空白孔 OD值 (0.563 g/l) μl 50 μl, Gonotec Osmomat 030,, 1 kg 1 mmol 1 (mosmol/kg) Na + -K + -ATP (NKA) 80 Na + -K + -ATP ( ), ( ) ATP ADP 1 μmol 1 ATP (U/mg prot) : Na + -K + -ATPase (U/mg prot)= 测定孔 OD值空白孔 OD值 6 7.8/ 标准孔 OD值空白孔 OD值 (mg prot/ml) (ALT), ALT, 510 nm OD, OD ( OD = OD OD ), ALT, (U/L) 1.3 EXCEL SPSS 13.0, ± ( x ±SD), (One-Way ANOVA) Duncan, P<0.05 2, 24,,,,, 2.1 1, Na +,, Na + (215.69±9.83) mmol/l, d, Na +, 18U (131.15± 14.42) mmol/l (P<0.05), (209.58±5.85) mmol/l 30 18, K + (P<0.05), h, K +, 18D 18U (P>0.05),, (7.97±0.20) mmol/l (8.21±0.38) mmol/l (P>0.05), Cl, 1, 30D 30U 24D 24U 18D 18U, (P>0.05) Cl Ca 2+ (3.78±0.49) mmol/l,, Ca 2+,, Ca 2+ (6.28±0.85) mmol/l, (P<0.05)

4 : 669 ion Tab. 1 表 1 低盐胁迫对仿刺参体腔液各离子浓度的影响 Effects of low salinity on the variations of ionic concentrations of sea cucumber n=3; x ±SD; mmol L 1 sampling stage 30D 24D 18D 18U 24U 30U Na + 215.69±9.

4 4 : 669 ion Tab. 1 表 1 低盐胁迫对仿刺参体腔液各离子浓度的影响 Effects of low salinity on the variations of ionic concentrations of sea cucumber n=3; x ±SD; mmol L 1 sampling stage 30D 24D 18D 18U 24U 30U Na ±9.83 d ±4.15 bc ±5.94 b ±14.42 a ±12.96 bcd ±5.85 cd K ±0.38 b 6.13±0.15 a 6.08±0.24 a 6.09±0.48 a 7.16±0.58 ab 7.97±0.20 b Ca ±0.49 a 4.87±0.96 ab 4.38±0.35 ab 4.40±0.32 ab 5.92±0.31 b 6.28±0.85 b Cl ±1.38 c ±1.38 b ±2.13 a ±3.04 a ±2.68 b ±3.70 c : (P<0.05). 30D: (31.09±0.12) ; 24D: (31) 24 ( 6 h 3 ) 6 h ; 18D: (31) 18 ( 6 h 3 ) 6 h. Note: Data with different letters in the same row indicate significant differences (P<0.05). 30D: coelomic fluid at salinity (31.09±0.12); 24D: sampling 6 h later after salinity declining from 31 to 24 (declining rate at 3/6 h); 18D: sampling 6 h after salinity declining from 31 to 18 (declining rate at 3/6 h). 2.2 ( 2), (910.67±15.34) mosmol/l 892 mosmol/l,, 18D (520.67±18.67) mosmol/l; 18U 466 mosmol/l 24 30, 2 (P<0.05). 30D: (31.09±0.12) ; 24D: (31) 24 ( 6 h 3 ) 6 h ; 18D: (31) 18 ( 6 h 3 ) 6 h. Fig. 2 The osmotic pressure of the rearing water and the coelomic fluid in Apostichopus japonicas during the period of low salinity change. Histogram bars with different letters indicate significant differences (P<0.05). 30D: coelomic fluid at salinity (31.09±0.12); 24D: sampling 6 h later after salinity declining from 31 to 24 (declining rate at 3/6 h); 18D: sampling 6 h after salinity declining from 31 to 18 (declining rate at 3/6 h). (682.67±2.19) mosmol/l (870±6.93) mosmol/l 30U, 875 mosmol/l (P<0.05), 2.3 Na + -K + -ATP Na + -K + -ATP 3 24 h Na + -K + -ATP, h, Na + -K + -ATP (P< 0.05), (1.39±0.19) U/mg prot, Na + -K + - ATP (0.24±0.03) U/mg prot,, Na + -K + -ATP, (0.91±0.08) U/mg prot 4 (1.01±0.10) gprot/l 24, (P<0.05), h, (0.45± 0.05) gprot/l, 18D (P>0.05),, (0.71±0.07) gprot/l 2.4, (10.13±0.77) ~ (20.71±4.22) U/L, (P >0.05)( 5)

5 Na + -K + -ATP (P<0.05). 30D: (31.09±0.12) ; 24D: (31) 24 ( 6 h 3 ) 6 h ; 18D: (31) 18 ( 6 h 3 ) 6 h. Fig. 3 The effect of acute low salinity stress on the activities of Na + -K + -ATPase in the coelomic fluid of Apostichopus japonicus Histogram bars with different letters indicate significant differences (P<0.05). 30D: coelomic fluid at salinity (31.09±0.12); 24D: sampling 6 h later after salinity declining from 31 to 24 (declining rate at 3/6 h); 18D: sampling 6 h after salinity declining from 31 to 18 (declining rate at 3/6 h). 4 (P<0.05). 30D: (31.09±0.12) ; 24D: (31) 24 ( 6 h 3 ) 6 h ; 18D: (31) 18 ( 6 h 3 ) 6 h. Fig.4 The effect of acute low salinity stress on the concentration of total protein in the coelomic fluid of Apostichopus japonicus Histogram bars with different letters in the same row indicate significant differences (P<0.05). 30D: coelomic fluid at salinity (31.09±0.12); 24D: sampling 6 h later after salinity declining from 31 to 24 (declining rate at 3/6 h); 18D: sampling 6 h after salinity declining from 31 to 18 (declining rate at 3/6 h) , 5 30D: (31.09±0.12) ; 24D: (31) 24 ( 6 h 3 ) 6 h ; 18D: (31) 18 ( 6 h 3 ) 6 h. Fig. 5 The effect of acute low salinity stress on the activities of GPT in the coelomic fluid of Apostichopus japonicus 30D: coelomic fluid at salinity (31.09±0.12); 24D: sampling 6 h later after salinity declining from 31 to 24(declining rate at 3/6 h); 18D: sampling 6 h after salinity declining from 31 to 18 (declining rate at 3/6 h)., [19] [20], 20~35, 25~ 30 18, Dong [21] Wang [5],,,,,, (Na + K + Cl )

6 4 : 671, [22], [23] Na + Cl, [24] (Onchidium struma) [1] (Rachycentron canadum Linnaeus), K +, Sampaiola [25] (Paralichthys orbignyanus) K +, K + Na + [26], K + [27],, K +, K + Na + Cl,, K + Ca 2+, [28] Ca 2+, (Dunaliella salina),,, Ca 2+ [29] Ca 2+, Ca 2+,, Ca 2+, Ca 2+,, Ca 2+ Ca 2+,, / Na + K + Ca 2+ [30] 3.2 Na + -K + -ATP Na + -K + -ATP, Na + K +, [22] [3] (Acipenser gueldenstaedtii) [29] Na + -K + -ATP,,, Na + -K + - ATP,, [2], Na + - K + -ATP [24], Na + -K + -ATP, [30],,, ;, 24 h, 18, Na + -K + - ATP, Na + -K + -ATP,, 18 3 d, Na + -K + -ATP, 18 Na + -K + -ATP,,, Na + -K + -ATP, Na + -K + -ATP,,,,

7 ,,, [4],, 2~5,, 18 24U,,,, 参考文献 : [1] Xu L W, Feng J, Guo Z X, et al. Effect of salinity on hematology and gill Na + -K + ATPase activity of juvenile cobia, Rachycentron canadum Linnaeus[J]. Marine Environmental Science, 2008, 27(6): [,,,. Na + -K + -ATPase [J]., 2008, 27(6): ] [2] Yu D G, Yang Y Q, Wang H Y, et al. The effect of salinity change on physiology and biochemistry of Epinephelus coioides[j]. Journal of Fisheries of China, 2011, 35(5): [,,,. [J]., 2011, 35(5): ] [3] Zhao F, Zhuang P, Zhang L Z, et al. The influence of salinity acclimation on activity of Na + /K + -ATPase in branchial epithelium, concentration of ions and osmolarity in serum of Acipenser schrenckii[j]. Journal of Fisheries of China, 2006, 30(4): [,,,. Na + -K + -ATP [J]., 2006, 30(4): ] [4] Liu W, Zhi B J, Zhan P R, et al. Effects of salinity on haematological biochem istrical indices and liver tissue in juvenile Oncorhynchus keta[j]. Chinese Journal of Applied Ecology, 2010, 21(9): [,,,. [J]., 2010, 21(9): ] [5] Wang Q L, Yu S S, Qin X C, et al. Combined effects of acute thermal and hypo-osmotic stresses on osmolality and hsp70, hsp90 and sod expression in the sea cucumber Apostichopus japonicas Selenka[J]. Aqua Intern, 2014, 22(3): [6] Wang J Q, Zhang Y X, Jiang Y S, et al. Effects of abrupt decline in salinity on growth, immunity and histological structure of respiratory trees in juvenile sea cucumber Apostichopus japonicas[j]. Journal of Dalian Fisheries University, 2009, 24(5): [,,,. [J]., 2009, 24(5): ] [7] Dong X L, Li C L, Zhao B, et al. Effecs of low salinity stress on the actinities of non-specific immune enzymes and antibacterial activity of sea cucumber Apostichopus japonicus[j]. Progress in Fishery Sciences, 2013, 34(3): [,,,. [J]., 2013, 34(3): ] [8] Tian D. Study on immune activity of sea cucumber (Apostichopus japonicus)in different weight and salinity[d]. Qingdao: Ocean University of China, 2013: [. (Apostichopus japonicus) [D]. :, 2013: 1 84.] [9] Yuan X T, Yang H T, Zhou Y, et al. Salinity effect on respiration and excretion of sea cucumber Apostichopus japonicus[j]. Oceanologia et Limnologia Sinica, 2006, 37(4): [,,,. [J]., 2006, 37(4): ] [10] Xue S Y, Fang J G, Mao Y Z, et al. Effects of salinity on the respiration and ammonia excretion of sea cucumber Apostichopus japonicus under high temperature[j]. Journal of Fishery Sciences of China, 2009, 16(6): [,,,. 1 [J]., 2009, 16(6): ] [11] Bao J, Jiang H B, Dong S L, et al. Comparison of oxygen consumption rate and ammonia-n excretion rate between green type and red type Apostichopus japonicus[j]. Journal of Fisheries of China, 2013, 37(11): [,,,. [J]., 2013, 37(11): ] [12] Wang C, Tian Y, Chang Y Q. Effect of salinity stress on immune enzyme activity of sea cucumber ( Apostichopus japonicus)[j]. Journal of Agricultural Science and Technol-

8 4 : 673 ogy, 2013, 15(3): [, 燚,,. [J]., 2013, 15(3): ] [13] Herrera F C, Herrera M I, López I. Further studies on the partial double Donnan: Is isosmotic KCl solution isotonic with cells of respiratory trees of the holothurian Isostichopus badionotus Selenka[J]. Journal of Experimental Marine Biology and Ecology, 2000, 247: [14] Diehl W J. Osmoregulation in echinoderms [J]. Comparative Biochemistry and Physiology Part A, 1986, 84(2): [15] Yancey P H, Clark M E, Hand S C., et al. Living with water stress: evolution of osmolyte systems[j]. Science, 1982, 217: [16] Meng X L, Dong Y W, Dong S L, et al. Mortality of the sea cucumber, Apostichopus japonicus Selenka, exposed to acute salinity decrease and related physiological responses: Osmoregulation and heat shock protein expression[j]. Aquaculture, 2011, 316(1 4): [17] Zheng H, Li B, Rong X J, et al. Effects of salinity and dissolved oxygen variation on the non-specific immune response of Apostichopus japonicus[j]. Progress in Fishery Sciences, 2014, 33(1): [,,,. [J]., 2014, 33(1): ] [18] Zang Y Q. Ecological and physiological mechanisms on typical environmental factors in sea cucumber, Apostichopus japonicus Selenka[D]. Qingdao: Ocean University of China, 2012: [. (Apostichopus japonicus Selenka) [D]. :, 2012: ] [19] Chen Y, Gao F, Liu G S, et al. The effects of temperature, salinity and light cycle on the growth and behavior of Apostichopus japonicus[j]. Journal of Fisheries of China, 2007, 31(5): [,,,. [J]., 2007, 31(5): ] [20] Gong H B, Wang Y B, Deng H, et al. Tolerance of sea cucumber (Apostichopus japonicus) to salinity[j]. Fisheries Science, 2009, 28(5): [,,,. [J]., 2009, 28(5): ] [21] Dong Y W, Dong S L, Meng X L. Effects of thermal and osmotic stress on growth, osmoregulation and Hsp70 in sea cucumber (Apostichopus japonicus Selenka)[J]. Aquaculture, 2008, 276(1 4): [22] Wang M L, Li C, Yang M, et al. Effects of salinity on osmotic adjustment of sea cucumber[j]. Fishery Modernization, 2014, 41(1): 6 9. [,,,. Na + -K + -ATP [J]., 2014, 41(1): 6 9.] [23] Chen C H, Zhou T, Yun H, et al. Research Advances on Voltage-gated Sodium Channels Disease[J]. Progress in Modern Biomedicine, 2013, 30(13): [,,,. [J]., 2013, 30(13): ] [24] Shen Y L, Ge X P, Huang J T, et al. Effects of salinity on Na + /K + -ATPase activity, the osmolality of pericardial cavity fluid and peritoneal fluid and ion content in Onchidium struma[j]. Journal of Fisheries of China, 2013, 37(6): [,,,. Na + / K + -ATP [J]., 2013, 37(6): ] [25] Sampaio L A, Bianchini A. Salinity effects on osmoregulation and growth of the euryhaline flounder Paralichthys orbignyanus[j]. J Exp Mar Biol Ecol, 2002, 269: [26] Zhang L Y. Effects and the osmoregulation mechanisms of calcium and magnesium concentrations and salinity acclimatization on serum osmolality of juvenile Paralichthys olivaceus[d]. Qingdao: Ocean University of China, 2010: [. [D]. :, 2010: ] [27] Chen H, Chen S L, Jiang J G. Effect of Ca 2+ channel block on glycerol metabolism in Dunaliella salina under hypoosmoic and hyperosmotic stresses [J]. PLoS One, 2011, 6(12): e [28] Liu J S. Cell Information and Rgulation[M]. The Second Edition. Beijing: Peking Union Medical College Press, 2004: [. [M]. 2. :, 2004: ] [29] Qu L, Zhuang P, Zhang L Z, et al. Influence of salinity change on serum osmolality, ions and gill Na + /K + -ATPase activity in juvenile Russian sturgeon (Acipenser gueldenstaedtii)[j]. Journal of Fishery Sciences of China, 2010, 17(2): [,,,. Na + /K + -ATP [J]., 2010, 17(2): ] [30] Zhang C J, Shi Z H, Wang J G, et al. On salinity-related effects on osmoregulation mechanism in marine teleost[j]. Marine Fishries, 2013, 35(1): [,,,. [J]., 2013, 35(1): ]

9 Effects of acute low salinity stress on the physiological indices of Apostichopus japonicus GENG Chenfan, TIAN Yi, ZHANG Yupeng, CHANG Yaqing, SONG Jian Key Laboratory of Mariculture & Stock Enhancement in North China Sea, Ministry of Agriculture, Dalian Ocean University, Dalian , China Abstract: To investigate the adaptation mechanisms of sea cucumbers to low salinity environments, physiological indices were measured in the coelomic fluid of Apostichopus japonicus under acute low salinity stress. Test specimens weighed (16.93±3.08) g ( x ±SE) in body weight. Experiments simulated the salinity changes observed in culture ponds in the rainy season. Salinity was gradually lowered from 30 to 18 ppt at a rate of 0.5 ppt/h, where it was maintained for 96 h. Salinity was then increased back to 30 at the same rate, where it was maintained for 24 h. The osmotic pressure of the coelomic fluid was measured directly with a cryoscopic osmometer. Other physiological indices were measured using the corresponding test kits. The results showed that trends of variation in the osmotic pressure, total protein concentration, and Na +, K +, and Cl concentrations of the coelomic fluid were consistent with the changes in salinity. The close relationship of osmotic pressure and water salinity indicate that sea cucumbers osmoregulate their coelomic fluid to approximate levels of environmental salinity. Minimum concentrations of Na +, K + and Cl [(131.15±14.42) mmol/l, (6.0±0.24) mmol/l, and (141.76±2.13) mmol/l, respectively] occurred at 18 ppt. After salinity was returned to normal levels (30 ppt), the three indices showed no significant difference from the control group (P<0.05). Experimental Ca 2+ concentrations were maintained at higher levels than in the control group, which varied from (3.78±0.49) mmol/l to (6.28±3.69) mmol/l (mean±se). The Na + -K + -ATPase activity of the coelomic fluid over 4 d at 18 ppt was significantly higher than at other salinities (P<0.05), indicating activation of Na + -K + -ATPase activity under salinities of 18. This result demonstrated that the osmoregulatory system of the sea cucumber was activated in external environments of low salinity. Variation in Na + -K + -ATPase activity can alter membrane permeability to maintain the balance of osmotic pressure between the extra and intracellular environments. Compared with the control group, the total protein concentration in the coelomic fluid initially decreased, followed by an increase to a steady value that was then maintained for the duration of the experimental period. Glutamate pyruvate transaminase activity of the coelomic fluid, which can indirectly reflect the condition of protein metabolism, showed no significant change under differing salinities. This result indicated that changes in salinity had little effect on the respiratory metabolism of the sea cucumber and that sea cucumbers can adapt to changes in the external salinity. Osmolarity of the coelomic fluid was strongly dependent on the concentrations of Na +, K + and Cl. This experimental work on the adaptation of sea cucumbers to environmental salinity furthers understanding of sea cucumber physiology and ecology and will inform future work on the aquaculture of sea cucumbers. Key words: Apostichpus japonicus; low salinity; physiological indices; Na + -K + -ATPase; glutamate pyruvate transaminase Corresponding author: TIAN Yi. tianyi@dlou.edu.cn

32期

32期中国农学通报 2014,30(32):38-42 Chinese Agricultural Science Bulletin 中草药饲料添加剂对黄鳝性激素含量的影响 1,2 1,2 1,2 2 赵庆凯, 丁炜东, 邴旭文, 王建新 ( 1 南京农业大学无锡渔业学院, 江苏无锡 214081; 2 中国水产