聚β-羟基丁酸酯对中国明对虾抗WSSV能力的影响

Effects of poly-β-hydroxybutyrate on WSSV resistance in Fenneropenaeus chinensis

摘要:

为探索聚β-羟基丁酸酯（PHB）对感染白斑综合征病毒（WSSV）的水生动物存活率、病毒含量的影响，实验采用单因子浓度梯度法，对感染WSSV的中国明对虾投喂添加了不同浓度聚β-羟基丁酸酯（0.0%、0.5%、1.0%、2.5%、5.0%、10.0%）的饵料，统计相同时间点对虾死亡数量、存活率、相对免疫保护率（RPS），并利用real-time PCR测定死亡对虾体内病毒绝对含量。结果显示，PHB对中国明对虾存活率、平均存活时间及体内病毒拷贝数均有一定影响，主要表现在与对照组（0.0%）相比，随PHB浓度的升高，实验组对虾存活率和平均存活时间呈现先上升后下降趋势。各组平均存活时间为82.23、90.71、95.55、91.15、85.56及79.40 h，1.0%浓度组实验对虾平均存活时间与0.5%及2.5%浓度组无显著差异（P>0.05），但是显著高于其余各组（P<0.05）；各组累计死亡率均为100%。另外还发现各组平均病毒拷贝数为1.08×10⁷，1.15×10⁷，4.75×10⁷，1.27×10⁷，1.14×10⁷，3.29×10⁶个/ng DNA；对照组与1%浓度组病毒含量具有显著差异（P<0.05）。结果表明，PHB能提高中国明对虾抗WSSV的能力且1% PHB浓度为最适浓度。

关键词:中国明对虾;聚β-羟基丁酸酯;存活率;白斑综合征病毒;攻毒实验;实时荧光定量

Abstract:

This study was designed to explore the anti-WSSV influences of PHB in Chinese shrimp to provide a theoretical basis for the use of PHB in the aquaculture industry.This study used the single factor concentration gradient method.Fenneropenaeus chinensis were artificially infected with WSSV.The F.chinensis were also fed bait that contained different concentrations of PHB(0.0%, 0.5%, 1.0%, 2.5%, 5.0% and 10.0%);these concentrations were adjusted based on the weights of the shrimp at the beginning of the day and the number of shrimp in the tank.During the experimental period, the dead shrimp were collected and stored at -20 ℃, and the time of death and water temperatures were recorded.After the experiment, some of the shrimp were selected based on the death curve, and DNA was extracted.The shrimp survival rates for each treatment were determined daily, and the treatments were compared at each time point in terms of mean survival time, survival rates and relative percent survival(RPS)rates.Moreover, the absolute WSSV loads among the dead shrimp were determined by real-time PCR.The results indicated that the PHB influenced the survival rates, mean survival time and WSSV loads in the shrimp.Compared with the control group, the survival rates and mean survival time of the experimental groups exhibited downward trends following initial increases as the concentrations of PHB increased.The mean survival time of the groups treated with 0.0%, 0.5%, 1.0%, 2.5%, 5.0% and 10.0% PHB were 82.23, 90.71, 95.55, 91.15, 85.56 and 79.40 h, respectively.There were no significant differences in the mean survival time among the shrimp that were supplied with feed containing the 0.5%, 1% and 2.5% concentrations of PHB(P>0.05), but the mean survival time of the other groups were all significantly lower than that of the 1.0% PHB group(P<0.05).The mortality rates for all groups were ultimately 100%.Compared to the control group, the survival rates of the experimental groups were higher and the WSSV loads were lower in the groups in which the PHB concentration did not exceed 2.5% at the same time points.However, similar results were not observed at PHB concentrations above 2.5%.The mean RPS rates of each group were 0.0, 0.364 6, 0.509 4, 0.411 6, -0.150 9 and 0.144 2.There was a significant difference in the RPS rates of the control and 1.0% PHB group(P<0.05).The RPS of the 1.0% PHB group was the highest.When the quantity of WSSV reached the threshold to cause death, the shrimp began to die.During the experimental period, the numbers of WSSV copies for each group were 1.08×10⁷, 1.15×10⁷, 4.75×10⁷, 1.27×10⁷, 1.14×10⁷ and 3.29×10⁶ copies/ng DNA.There was a significant difference between the group that was supplied with 1.0% PHB and the control group(P<0.05).We also found that the numbers of WSSV copies in the 1.0% PHB treatment group were lower than those of the control group in the first 72 h(P<0.05), but higher 72 h later(P<0.05).These results indicated that PHB enhanced the immunity to and tolerance of WSSV in shrimp.This study also revealed that high concentrations of PHB inhibited shrimp survival.The present study found that 1% PHB in the feed was the preferable concentration for improving the ability of shrimp to resist WSSV.

Key words:Fenneropenaeus chinensis;poly-β-hydroxybutyrate;survival rate;WSSV;challenge test;real-time PCR