主要环境因子对养殖对虾抗病力及白斑综合症发生的影响
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摘要
白斑综合症病毒（white spot syndrome virus, WSSV）是已报道的所有对虾病毒中毒力最强、危害最大的一种病毒。对虾疾病的发生是机体自身的抗病力水平、病原微生物以及环境条件等多种因素相互作用和影响的结果。对疾病问题的研究必须从多个角度入手综合考虑。其中从环境角度入手，研究环境因子对对虾抗病力与病原生物致病力的影响是根本解决病害的重要对策之一。本文在国家973项目“环境胁迫对对虾抗病力的影响及其人工调控”的支持下，从诸多环境因子中选择温度（理化因子），氨氮（污染因子）及微藻（生物因子）为代表，从感染对虾的累计死亡率、病毒感染程度、对虾抗病力（血细胞密度THC、血清及血细胞酚氧化酶PO活力、血清碱性及酸性磷酸酶活力、血清蛋白质含量）等多个角度，研究了上述环境因子对对虾抗病力以及白斑综合症发生的影响。主要成果有：
1．温度对养殖对虾白斑综合症发生的影响 以人工感染的日本对虾为实验材料，比较了不同温度条件下白斑综合症发生的迟缓，体内THC、血清PO活力以及病毒感染程度的差别。结果显示温度是影响感染对虾发病死亡的重要因子，当养殖温度为15, 23, 28, 33℃时，各试验组的LT50分别为16.0, 4.5, 4.5, 9.0 d，说明温度影响感染WSSV的日本对虾发病的快慢，低温和高温条件下发病缓慢。各组病毒感染强弱依次为：28℃组> 23℃组> 33℃组> 15℃组，感染程度与LT50负相关。感染病毒2d后，各实验组对虾THC都低于对照组，其中15℃组最高，而28℃组最低。15℃组与33℃组PO活力有显著区别，而白斑综合症的暴发均变缓，说明低温和高温下白斑综合症暴发趋缓的机制可能存在差别。
2．温度突变对养殖对虾白斑综合症发生的影响 分别以感染WSSV的日本对虾和中国对虾为实验材料，研究了温度突变对对虾抗病力与白斑综合症发生的影响。对日本对虾进行温度突变，发现温度突变（24-27℃/6h;24-30℃/6h）不会引起健康对虾的死亡，可加速染病的日本对虾死亡。WSSV感染使日本对虾THC显著下降，温度突变加剧THC的下降，其下降幅度与感染时间，突变温度有关；通过低剂量感染中国对虾，造成温度恒定组不死亡，对虾处于潜伏感染状态，而温度变化可导致对虾发病死亡。累计死亡率主要与温度变化幅度有关，温度变化的速度影响对虾死亡的迟缓。温度变化可加剧感染WSSV 的中国对虾THC的下降，实验初
期血细胞PO活力升高，而后期血细胞PO活力降低。
3．氨氮胁迫对养殖对虾抗病力的影响 以THC、血细胞PO活力、血清蛋白质含量、碱性磷酸酶活力、酸性磷酸酶活力等为指标研究氨氮胁迫对健康对虾和感染WSSV的对虾抗病力的影响。氨氮胁迫可影响感染WSSV的对虾THC，血细胞PO活力以及血清蛋白质含量。氨氮对健康对虾THC，血细胞酚氧化酶活力，血清酸性磷酸酶和碱性磷酸酶均有影响。氨氮胁迫可降低健康的以及感染WSSV的对虾抗病力水平，应当采取有效措施降低虾池的氨氮浓度，以避免病害的发生。
4．微藻对对虾感染白斑综合症病毒的影响 养殖水体中一定浓度的赤潮异弯藻可延缓感染WSSV的中国对虾疾病的发生。以虾池常见微藻小球藻为实验材料，将小球藻分别与病毒粗提液、染病对虾共培养，通过套式PCR病毒检测技术检测初步证实小球藻可携带WSSV。
White spot syndrome (WSS) is the most virulent among the penaeid shrimp virus that had been reported and caused serious shrimp and economic losses. Disease is the final result of a complex interaction among the shrimp, its environment and the pathogen itself. To resolve the problem of disease must research from many aspects. Studies on the environmental factors on the immune responses of penaeid shrimp and the infectivity of WSSV is one of the strategies to control the disease radically. We selected some typical environmental factors including temperature, ammonia nitrogen and microalgae and studied the effect of these environmental factors on the immune responses and outbreaks of WSS in Marsupenaeus japonicus and Fenneropenaeus chinsis from these aspects containing cumulative mortality, infection level , immune responses of shrimp (using Total Haemocyte Counts, plasma and HLS phenoloxidase, plasma alkaline phosphatase and acid phosphatase, plasma protein concentrations as evaluation parameters). The main results are as follows，
1．Effects of temperature on the immune responses and outbreaks of WSS in penaeid shrimp The effects of temperature on outbreak of white spot syndrome of M. japonicus were studied through simulated experiments. The results indicated that outbreak of white spot syndrome in infected M. japonicus were relatively slow at 15℃and 33℃ and were fast at 23℃ and 28℃. The infected level were 28℃ group> 23℃ group > 33℃ group > 15℃ group. Accordingly, the experimental results showed that THC in infected M. japonicus were all lower than the control. With regard to PO activities, the value of 15℃ group was slightly lower than the control but that of 33℃ group was significantly higher than that. It was inferred that mechanisms of outbreak at
low and high temperatures could be different.
2．Effects of temperature acute changes on the immune responses and outbreaks of WSS in penaeid shrimp Temperature acute changes (24-27℃/6h; 24-30℃/6h) can not arise the death of healthy M. japonicus, but accelerate outbreak of WSS and death of shrimp infected with WSSV. THC of M.japonicus decreases after infection and temperature acute changes accelerate the decreasing process. The decreasing level was correlated to the infection time and the range of temperature acute changes. In the initial stages, HLS PO increased due to stimulation and decreased in the anaphase.
3．Effects of ammonia nitrogen on immune responses of healthy and infected penaeid shrimp Ammonia nitrogen can affect THC, HLS PO activities and plasma protein concentrations of M.japnicus infected with WSSV. It can also affect THC, HLS PO activities, plasma alkaline phosphatase and acid phosphatase of healthy F. chinesis. The results indicated that ammonia nitrogen could reduce the immune responses of healthy F.chinesis and infected M. japonicus. Effective measures should be carried out to reduce the concentration of ammonia nitrogen in the shrimp pond in order to improve the immune responses and be avoid of outbreaks of disease.
4．Effect of microalgae on outbreaks of white spot syndrome in penaeid shrimp Outbreaks of WSS in F.chinesis was slowed after introducing Heterosigma akashiwa and the introduced microalgae can weaken the infections of WSSV in F. chinesis. Chlorella was incubated with virus solution or infected M.japonicus, respectively. The primary results detected by nested-PCR method indicated that Chlorella could carry WSSV.
1．温度对养殖对虾白斑综合症发生的影响 以人工感染的日本对虾为实验材料，比较了不同温度条件下白斑综合症发生的迟缓，体内THC、血清PO活力以及病毒感染程度的差别。结果显示温度是影响感染对虾发病死亡的重要因子，当养殖温度为15, 23, 28, 33℃时，各试验组的LT50分别为16.0, 4.5, 4.5, 9.0 d，说明温度影响感染WSSV的日本对虾发病的快慢，低温和高温条件下发病缓慢。各组病毒感染强弱依次为：28℃组> 23℃组> 33℃组> 15℃组，感染程度与LT50负相关。感染病毒2d后，各实验组对虾THC都低于对照组，其中15℃组最高，而28℃组最低。15℃组与33℃组PO活力有显著区别，而白斑综合症的暴发均变缓，说明低温和高温下白斑综合症暴发趋缓的机制可能存在差别。
2．温度突变对养殖对虾白斑综合症发生的影响 分别以感染WSSV的日本对虾和中国对虾为实验材料，研究了温度突变对对虾抗病力与白斑综合症发生的影响。对日本对虾进行温度突变，发现温度突变（24-27℃/6h;24-30℃/6h）不会引起健康对虾的死亡，可加速染病的日本对虾死亡。WSSV感染使日本对虾THC显著下降，温度突变加剧THC的下降，其下降幅度与感染时间，突变温度有关；通过低剂量感染中国对虾，造成温度恒定组不死亡，对虾处于潜伏感染状态，而温度变化可导致对虾发病死亡。累计死亡率主要与温度变化幅度有关，温度变化的速度影响对虾死亡的迟缓。温度变化可加剧感染WSSV 的中国对虾THC的下降，实验初
期血细胞PO活力升高，而后期血细胞PO活力降低。
3．氨氮胁迫对养殖对虾抗病力的影响 以THC、血细胞PO活力、血清蛋白质含量、碱性磷酸酶活力、酸性磷酸酶活力等为指标研究氨氮胁迫对健康对虾和感染WSSV的对虾抗病力的影响。氨氮胁迫可影响感染WSSV的对虾THC，血细胞PO活力以及血清蛋白质含量。氨氮对健康对虾THC，血细胞酚氧化酶活力，血清酸性磷酸酶和碱性磷酸酶均有影响。氨氮胁迫可降低健康的以及感染WSSV的对虾抗病力水平，应当采取有效措施降低虾池的氨氮浓度，以避免病害的发生。
4．微藻对对虾感染白斑综合症病毒的影响 养殖水体中一定浓度的赤潮异弯藻可延缓感染WSSV的中国对虾疾病的发生。以虾池常见微藻小球藻为实验材料，将小球藻分别与病毒粗提液、染病对虾共培养，通过套式PCR病毒检测技术检测初步证实小球藻可携带WSSV。
White spot syndrome (WSS) is the most virulent among the penaeid shrimp virus that had been reported and caused serious shrimp and economic losses. Disease is the final result of a complex interaction among the shrimp, its environment and the pathogen itself. To resolve the problem of disease must research from many aspects. Studies on the environmental factors on the immune responses of penaeid shrimp and the infectivity of WSSV is one of the strategies to control the disease radically. We selected some typical environmental factors including temperature, ammonia nitrogen and microalgae and studied the effect of these environmental factors on the immune responses and outbreaks of WSS in Marsupenaeus japonicus and Fenneropenaeus chinsis from these aspects containing cumulative mortality, infection level , immune responses of shrimp (using Total Haemocyte Counts, plasma and HLS phenoloxidase, plasma alkaline phosphatase and acid phosphatase, plasma protein concentrations as evaluation parameters). The main results are as follows，
1．Effects of temperature on the immune responses and outbreaks of WSS in penaeid shrimp The effects of temperature on outbreak of white spot syndrome of M. japonicus were studied through simulated experiments. The results indicated that outbreak of white spot syndrome in infected M. japonicus were relatively slow at 15℃and 33℃ and were fast at 23℃ and 28℃. The infected level were 28℃ group> 23℃ group > 33℃ group > 15℃ group. Accordingly, the experimental results showed that THC in infected M. japonicus were all lower than the control. With regard to PO activities, the value of 15℃ group was slightly lower than the control but that of 33℃ group was significantly higher than that. It was inferred that mechanisms of outbreak at
low and high temperatures could be different.
2．Effects of temperature acute changes on the immune responses and outbreaks of WSS in penaeid shrimp Temperature acute changes (24-27℃/6h; 24-30℃/6h) can not arise the death of healthy M. japonicus, but accelerate outbreak of WSS and death of shrimp infected with WSSV. THC of M.japonicus decreases after infection and temperature acute changes accelerate the decreasing process. The decreasing level was correlated to the infection time and the range of temperature acute changes. In the initial stages, HLS PO increased due to stimulation and decreased in the anaphase.
3．Effects of ammonia nitrogen on immune responses of healthy and infected penaeid shrimp Ammonia nitrogen can affect THC, HLS PO activities and plasma protein concentrations of M.japnicus infected with WSSV. It can also affect THC, HLS PO activities, plasma alkaline phosphatase and acid phosphatase of healthy F. chinesis. The results indicated that ammonia nitrogen could reduce the immune responses of healthy F.chinesis and infected M. japonicus. Effective measures should be carried out to reduce the concentration of ammonia nitrogen in the shrimp pond in order to improve the immune responses and be avoid of outbreaks of disease.
4．Effect of microalgae on outbreaks of white spot syndrome in penaeid shrimp Outbreaks of WSS in F.chinesis was slowed after introducing Heterosigma akashiwa and the introduced microalgae can weaken the infections of WSSV in F. chinesis. Chlorella was incubated with virus solution or infected M.japonicus, respectively. The primary results detected by nested-PCR method indicated that Chlorella could carry WSSV.
引文
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