【作者】 程波；

【导师】 杨红生； 刘鹰；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生态学， 2010， 博士

【摘要】 本文以凡纳滨对虾封闭循环水养殖系统为研究对象,模拟养殖源水受Cu污染条件,研究Cu²⁺对养殖对虾和生物滤器的影响;查明系统中Cu的收支、分布和代谢途径;并从养殖水环境、对虾体液免疫、消化功能、组织损伤、生理代谢、重金属富集等方面,比较Cu污染对凡纳滨对虾在封闭循环水养殖系统和静水养殖系统中的影响程度。主要研究结果如下:1.查明了Cu对凡纳滨对虾稚虾（体长4.0±0.4cm,体重0.94±0.02g）在24h、48h、72h和96h的半致死浓度(LC₅₀)分别为54.81mg/L、42.92mg/L、23.91 mg/L和13.11mg/L。Cu²⁺胁迫能破坏凡纳滨对虾鳃丝的组织结构和功能,抑制耗氧速率和鳃丝中Na⁺-K⁺-ATP酶活性,诱导肝胰腺中金属硫蛋白（MT）的合成和排氨率的增加,引起对虾死亡。2.凡纳滨对虾（体长8.5±0.76cm,体重7.6±0.5g）分别暴露于0.5mg/L和5mg/L Cu²⁺浓度7天,耗氧率分别降低24.62%和44.03%,排氨率分别增加78.26%和175.69%;Cu能在凡纳滨对虾体内富集,各组织中富集量顺序依次为:肝胰腺>鳃>甲壳>肌肉,肝胰腺中富集量约为肌肉中富集量的100倍。3.质量浓度为0.3mg/L的Cu²⁺浓度污染,对循环水养殖系统生物滤器中的异养细菌（HB）、氨氧化菌（AOB）和亚硝酸盐氧化菌（NOB）数量影响不显著,对生物滤器的COD、NH₄-N和NO2-N去除率影响不显著。养殖实验初期,填料表面HB、AOB和NOB数量级分别为103 CFU/g、102-103 MPN/g和100-101 MPN/g;养殖过程中,三类细菌数量均呈现逐渐增加的趋势;在生物滤器稳定运行阶段,HB数量级为106 CFU/g,AOB和NOB数量级为104-105 MPN/g。HB和AOB数量达到稳定约需30 d,NOB约需45 d。养殖过程中生物滤器对COD、NH₄-N和NO₂-N平均去除率分别为10%、18.5%和29%。4.质量浓度为0.3mg/L的Cu²⁺污染对封闭循环水养殖系统的运行不造成显著影响。处理组和对照组相比,在对虾体重（11.08±0.76 mg vs 10.52±0.51 mg）,产量（6.42±0.33 kg/m³ vs 5.99±0.39 kg/m³）,存活率（67.78±5.29 % vs 66.39±1.93 %）和饵料转化率FCR（1.2±0.060 vs 1.29±0.082）方面差异不显著。养殖过程中对照组NH₄-N和NO₂-N的平均浓度分别为0.28±0.13mg/L和4.02±5.60mg/L,处理组中分别为0.26±+0.1 mg/L和4.42±6.09mg/L。封闭循环水养殖系统能够提供较好的水质条件,获得较高的产量和较低的FCR。5.在质量浓度为0.3mg/L的Cu²⁺污染条件下,输入水体中的Cu是封闭循环水养殖系统中Cu的主要来源,占全部Cu来源的75.69%,其次是投喂饲料,占全部Cu来源的21.03%。在系统中,机械过滤和排污分别排出全部Cu总量的41.72%和15.05%,养殖水体中Cu²⁺浓度在养殖结束时降为0.089±0.012 mg/L,对凡纳滨对虾体液免疫指标、消化酶活等影响不显著。所养对虾体内Cu富集含量为42.91±6.10 mg/Kg,显著增加,但仍在国家“无公害食品水产品中有毒有害物质限量”农业行业标准（NY 5073-2006）范围之内（≤50 mg/Kg）。6.在无Cu²⁺污染的对虾封闭循环水养殖系统中,投喂饵料是系统中Cu的主要来源,占全部来源86.55%。系统中Cu主要通过机械过滤、排水和排污三大途径排放到系统外,分别排放Cu总量的22.33%、12.43%和11.83%。加强机械过滤单元的设计与管理,是减少Cu²⁺对系统造成污染的关键。在98 d的对虾养殖实验中,通过过滤、排水和排污等方式分别排出14.7 mg、7.94 mg和7.56 mg的Cu。7.在质量浓度为0.8mg/L Cu²⁺污染条件下,封闭循环水养殖系统与静水养殖系统相比,在对虾存活率（40.00±2.22 % vs 15.44±3.07 %）、体重（13.36±0.74 g vs 10.34±1.15 g）和FCR（2.03±0.12 vs 6.52±0.32）上差异显著,养殖过程中水体NH4-N（1.63±1.15 mg/L vs 6.48±8.41 mg/L）、NO2-N（1.03±0.39 mg/L vs 6.09±1.32 mg/L）和COD（10.93±4.80 mg/L vs 24.36±13.87 mg/L）平均浓度差异显著。相较于静水养殖系统,封闭循环水养殖系统能够提供更好的水质条件,获得更快的生长速度和更低的FCR。8.在质量浓度为0.8mg/L Cu²⁺污染条件下,封闭循环水养殖系统和静水养殖系统中对虾血淋巴SOD、酚氧化酶活性和大颗粒细胞数量、血细胞总数和胃中淀粉酶活性均显著降低,但在静水系统中,酚氧化酶活性（0.008±0.003U/min/mg pr vs 0.011±0.007 U/min/mg pr）受抑制程度显著增加。在免疫基因表达层面,封闭循环水养殖系统中对虾SOD mRNA相对表达量为1.41±0.57,显著增加,通过基因表达量增加对蛋白水平酶活性丧失进行补偿;溶菌酶和酚氧化酶基因mRNA相对表达量增加为1.49±1.32和1.40±1.19,差异不显著。静水养殖系统中,SOD和溶菌酶基因mRNA相对表达量为0.71±0.42和0.64±0.53,受到显著抑制;酚氧化酶基因mRNA相对表达量为1.64±1.12,显著增加,通过基因表达量增加对蛋白水平酚氧化酶活性丧失进行补偿。可见,该浓度Cu²⁺在两种养殖模式中均能降低凡纳滨对虾体液免疫水平,且在静水养殖系统中影响程度更大;Cu²⁺胁迫下,凡纳滨对虾具有通过调节基因表达水平进而对蛋白酶活性损失进行补偿的能力;相较于酚氧化酶基因,SOD和溶菌酶基因对Cu²⁺的胁迫更为敏感,且表达更易受到抑制。9.在质量浓度为0.8mg/L的Cu²⁺污染条件下,在封闭循环水养殖系统和静水养值系统中,对虾生理代谢、鳃丝中Na+-K+- ATP酶活性均受到显著抑制,并致使鳃丝柱细胞和上皮细胞丢失,结构扭曲,甚至失去完整的组织结构;以及肝胰腺肌上皮层与上皮细胞的分离,储存细胞与分泌细胞部分丢失,组织间隙水肿等症状,对鳃丝和肝胰腺的组织结构和功能均造成显著破坏和影响。对虾肝胰腺中金属硫蛋白（MT）含量分别为2.27±0.62nmol/g和2.75±0.55nmol/g,具有显著差异;Cu的富集含量分别为834.52±267.41mg/kg干重和1398.31±263.90mg/kg干重,具有显著差异。MT含量对Cu²⁺胁迫敏感,并与肝胰腺中Cu的富集含量呈现较好的剂量效应关系,可望在量化封闭循环水养殖系统和静水养殖系统中对虾受胁迫程度的比较研究中得以应用。更多还原

【Abstract】 The purpose of present study was to investigate the performance of Litopenaeus. vannamei recirculating aquaculture system （RAS） under copper pollution , the effect of copper on bacteria community in biological aerated filter （BAF） and shrimp, and the copper budget, distribution and emission facters in the shrimp RAS. Furthermore, under the same copper concentration in both RAS and static aquaculture system, comparing the difference of shrimps’immunity, digestive tract, histological changes, physiological metabolism and heavy metal accumulation was also covered in the study. The main results include:1. The Medium Lethal Concentration (LC₅₀) of Cu to Litopenaeus. vannamei （body length was 4.0±0.4cm, weight was 0.94±0.02g） was evaluated and the study showed that 24h LC₅₀, 48h LC₅₀ , 72h LC₅₀ and 96h LC₅₀ of Cu was 54.81mg/L, 42.92mg/L, 23.91 mg/L and 13.11mg/L respectively. Acute toxicity of Cu²⁺ can cause death , MT production in hepatopancreatic, ammonium excretion increase, inhibition in oxygen consumption rate and activity of Na+-K+-ATPase within gills of shrimp, however, there was no evidence to illustrate that molting behavior was also result from it.2. After Litopenaeus. vannamei （body length was 8.5±0.76cm, weight was 7.6±0.5g） were exposed in 0.5mg/L and 5mg/L Cu²⁺ solution for 7 days, oxygen consumption rate decreased by 24.62% and 44.031% and ammonium excretion was elevated by 78.26% and 175.69%, respectively. The study showed that copper can be accumulated within hepatopancreas, gills, carapaces and muscles increasingly, and the amount of accumulated Cu in hepatopancreas was 100 times higher than it in muscles. So it was sufficient to conclude that Cu accumulation was most significant in hepatopancreas out of the four organs in the study for Litopenaeus. vannamei in the specific life conditions as we had.3. Three months’experiment was conducted for intensive culture of shrimp （L. vannamei） in RAS to study the effect of Cu polluted water (0.3mgL^-1) on the biofilm. The results indicated that there was no significant difference in COD, NH₄-N and NO₂-N removal rate between regular RAS system and the one worked with biofilm. Also the number of Heterotrophic Bacteria（HB）, Ammonium-oxidation bacteria（AOB） and Nitrite-oxidizing bacteria（NOB） in the biofilm was not effected dramatically. The number of Heterotrophic Bacteria, Ammonium-oxidation bacteria and Nitrite-oxidizing bacteria were only 103, 102-103 and 100-101 per gram packing at the beginning, and it took 30 days for the number of Heterotrophic Bacteria and Ammonium-oxidation bacteria to reach the stable level, which were 106 and 104-105 per gram packing respectively. But for Nitrite-oxidizing bacteria, it took nearly 45 days to reach the stable number of 104-105 per gram packing.4. Harvesting was obtained after 98 days’culture, and it showed that there was no significant difference in mean body weight （11.08±0.76 mg vs 10.52±0.51 mg）, yield （6.42±0.33 kg/m³ vs 5.99±0.39 kg/m3）, survival rate （67.78±5.29 % vs 66.39±1.93 %） and FCR （1.2±0.060 vs 1.29±0.082） between the Cu polluted systems and the control systems （P>0.05）. Shrimp growth was not limited by any of the water quality parameters in the study. TAN and NO2-N concentrations were 0.28±0.13mg/L and 4.02±5.60mg/L in the control systems and the values were 0.26±+0.1 mg/L and 4.42±6.09mg/L respectively. Juveniles of the Pacific white shrimp, L. vannamei, can be raised in limited water exchange regime with healthy conditions, low feed conversion rate, fast growth, and high biomass loading in RAS.5. Cu concentration decreased from 0.3 mg/L to 0.089±0.012mg/L at the harvesting time and most of the Cu had been removed out from the water treatment processes. The major output of Cu was in the mechanical filter and the draining effluent in the capture, which accounted for 41.72% and 15.05% of the total inputs respectively. As a result, the cultured shrimp had not been effected on immunity, digestive tract with Cu accumulation in tissues. The molality of Cu in the shrmps was at last 42.91±6.10 mg/Kg, and it meet the requirment of the Green Food and Pollution-Free Agriculture Products Standard, which gave the molality limitation as≤50 mg/Kg.6. Cu budget in the shrimp RAS revealed that the most source of Cu to the systems was the aqua feeds, which accounted for 86.55% of the total inputs. The major output of Cu was in the mechanical filter, renewal waters and the draining effluent during the capture activity, which accounted for 22.33%, 12.43% and 11.83% of the total inputs respectively. Another, Cu loading releasing from the three ways was 14.7 mg /cycle, 7.94 mg/ cycle and 7.56 mg /cycle respectively.7. Harvesting was also obtained after 112 days culture, and there was significant difference in mean survival rate （40.00±2.22 % vs 15.44±3.07 %）, body weight （13.36±0.74 g vs 10.34±1.15 g） and FCR （2.03±0.12 vs 6.52±0.32） between the RAS and the static aquaculture system under 0.8 mg/L of copper pollution （P>0.05）. The mean concentrations of TAN （1.63±1.15 mg/L vs 6.48±8.41 mg/L）, NO₂-N （1.03±0.39 mg/L vs 6.09±1.32 mg/L） and COD （10.93±4.80 mg/L vs 24.36±13.87 mg/L） were also significant to each other in the two systems. Juveniles of the Pacific white shrimp, L. vannamei, had good aqueouse environment achieved faster growth rate with lower feed conversion rate in RAS comparing to those in static aquaculture system.8. PO activity, SOD activity, quantity of large granular cells and total haemocyte count of Litopenaeus vannamei had significantly decreased after shrimps been cultured for 112 days with 0.8mg/L copper molality in both RAS and static aquaculture system. The result showed that PO activity （0.008±0.003U/min/mg pr） of shrimp in static aquaculture system was significantly lower compared to in RAS （0.011±0.007 U/min/mg pr）. In RAS, SOD gene expression levels of shrimp was 1.41±0.57, and it had been elevated very much; mRNA levels of Lys and ProPO increased to 1.49±1.32 and.40±1.19 respectively but no significant differences were detected. In static aquaculture system, SOD and Lys gene expression levels of shrimp were dramatically decreased to 0.71±0.42 and 0.64±0.53, however, the mRNA level of ProPO significantly increased to 1.64±1.12. The upregulation of proPO mRNA by the shrimp may be resulted in decreased PO activity. The present study documented that the concentration of Cu²⁺ at 0.8mg/L decreased the humoral immunity of Litopenaeus vannamei both in RAS and static aquaculture system, the Litopenaeus vannamei can regulate the expression levels of gens to banlance the decreased enzyme activity as immune modulation to enhance the immune ability. In addition, the mRNA transcription of SOD and Lys gen in shrimp is sensitivity to the copper pollution compared to proPO gen.9. The toxicity of Cu²⁺ at 0.8mg/L inhibited oxygen consumption, Na⁺-K⁺- ATPase activity and elevated in ammonium excretion in Litopenaeus vannamei both in RAS and static aquaculture system. The gills of shrimps collected in static aquaculture system after been cultured in 0.8 mg/L copper pollution water for 112 days showed a high disorder and a high degree of melanization. Absence of pillar cells, loss of regular structure of epithelium and multifocal necrosis on gills of shrimp were observed in the RAS. To the histological changes of hepatopancreas, atrophy and irregular tubular structure were observed in shrimps collected in static aquaculture system at the harvest. In the RAS, there was an absence of storage and secretory cells, as well as multifocal necrosis and loss of tissue in some zones of the tubules, separation between myoepithelium and epithelium.The amount of MT induced in the hepatopancreas was 2.27±0.62nmol/g and 2.75±0.55nmol/g respectively in RAS and static aquaculture system, and the Cu concentration accumulated in hepatopancreas was 834.52±267.41mg/kg dry weight and 1398.31±263.90mg/kg dry weight respectively, which showed a significant difference with each other. There was significant linear correlation between the Cu concentration and MT, Which may be used as a valid biomarkers to compared the RAS and static aquaculture system under heavy metal pollution.更多还原