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苯并[a]芘(Benzo(a) pyrene,BaP)是美国环保局(EPA)优先检测的16种多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)中危害最大的一种[1]。其分布广泛,致癌性强且化学性质稳定,可通过地表径流、污水排放、海洋倾废及大气沉降等途径输入海洋环境中,对海洋生态系统和人类健康构成了极大的威胁[2-5]。研究表明,BaP可以在珊瑚体内富集,导致珊瑚虫NADPH色素P450 C减少[6],影响珊瑚的正常生长发育,扰动或破坏珊瑚与虫黄藻的共生关系,从而导致珊瑚白化[7-8]。2015年海南省洋浦湾表层沉积物中总的PAHs质量分数达到158 3.2~570 1.7 µg·kg−1,海口湾海水PAHs的质量浓度达到420.2~2 539.1 ng·L−1,污染程度已达中等水平,具有潜在的生态风险[9-10]。此外,XIANG等[11]的研究显示,由于生物蓄积,珊瑚中PAHs浓度明显高于周围沉积物,环境中的PAHs已经对珊瑚的健康产生了一定的影响。因此,针对BaP对珊瑚−虫黄藻共生体的胁迫研究,对保护南海珊瑚礁生态系统具有重要意义。
珊瑚礁是海洋生态系统的重要组成部分,然而由于人类活动和自然环境的变化,全球珊瑚礁生态系统正面临日益严重的退化趋势[12]。造礁石珊瑚和虫黄藻的共生关系是珊瑚礁生态系统的重要基础[13],虫黄藻生活于珊瑚内胚层细胞质中,受到珊瑚与虫黄藻形成的一种共生体结构的保护[14]。虫黄藻对珊瑚至关重要,它通过珊瑚虫获得代谢产物CO2、N和P进行光合作用,同时为珊瑚虫提供溶解氧和有机物如葡萄糖、甘油、氨基酸等,促进珊瑚的生长[15-17]。但是,当环境条件发生剧烈变化时,例如温度上升,环境污染物胁迫等,会影响虫黄藻与珊瑚的共生关系,从而导致珊瑚白化[18]。
珊瑚白化是一种重要的生理现象,会导致珊瑚钙化速率与生长速度降低、繁殖力下降、疾病增多和大量死亡,最终导致整个珊瑚礁生态系统功能与多样性的退化[19]。当发展到肉眼可见白化现象时,珊瑚体内的虫黄藻己经损失了70%~90%;当环境因子的变化超过珊瑚虫的耐受范围,虫黄藻的光合器官受到损伤,导致光合效率下降[20]。珊瑚白化一段时间后,如果无法恢复正常或者应激持续时间过长,将会导致珊瑚死亡[21]。在珊瑚白化过程中,虫黄藻的呼吸效率、密度以及叶绿素a的含量都有可能改变[22]。因此,维持珊瑚−虫黄藻的共生关系对于珊瑚礁生态系统的恢复力和耐受性都至关重要[23]。VAN等[24]提出协同进化提高珊瑚适应性的理论,强调培育环境耐受性珊瑚的重要性。ANTHONY等[25]主张将协同基因流、协同进化、系统生物学、栖息地工程化保护和污染物控制联合应用到未来珊瑚礁的保护之中。因此,本研究针对BaP胁迫时3种不同属珊瑚(Pocillopora damicornis,Montipora digitata,Acropora formosa)的光合与呼吸效率、密度以及叶绿素a的含量这些生理指标的变化,探究不同种属珊瑚对PAHs类污染物胁迫的敏感性,不仅可以增强人们对于珊瑚−虫黄藻共生关系的理解,还可以为选育环境耐受性珊瑚种质提供重要参考。
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由图1可知,对于同一种珊瑚,以0天为对照,随着胁迫时间的增加叶绿素a含量整体呈下降趋势。与0天相比,指状蔷薇珊瑚在12.5 μg·L−1时叶绿素含量差异不显著(P>0.05),而鹿角杯型珊瑚和美丽鹿角珊瑚差异显著(P< 0.05)。3种珊瑚在胁迫7 d时,叶绿素a含量下降的变化达极显著(P <0.01)。由图2可知,与0 μg·L−1的DMSO对照组相比,3种珊瑚叶绿素a含量在胁迫后都出现显示出了下降趋势,其中,美丽鹿角珊瑚分别在100 μg·L−1胁迫5 d和50 μg·L−1胁迫7 d时,叶绿素a含量接近0 mg·L−1。3种珊瑚随着胁迫浓度的增加叶绿素a含量显著下降,同一胁迫浓度下,时间越久叶绿素a含量下降越显著。其中,指状蔷薇珊瑚的叶绿素a变化程度较低,表现出较强的耐受性,美丽鹿角珊瑚对BaP胁迫敏感。
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共生虫黄藻密度的变化与叶绿素a含量的变化显现出相似趋势。t检验差异显著(P < 0.05)。由图3和图4可知,美丽鹿角珊瑚的共生虫黄藻密度减少更严重。在100 μg·L−1胁迫5 d时,美丽鹿角珊瑚的虫黄藻密度降至(0.6287 ± 0.2489)×104 个·cm−2,此时已经严重白化;在50 μg·L−1胁迫7 d时,美丽鹿角珊瑚的虫黄藻密度降到(0.5297 ± 0.1465)×104 个·cm−2,鹿角杯型珊瑚在100 μg·L−1胁迫7 d后,虫黄藻密度下降到最低水平[(6.3726 ± 3.9607)×104 个·cm−2],而相同胁迫条件下的指状蔷薇珊瑚与虫黄藻的共生关系则显示出更强的稳定性。
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本研究中使用GraphPad Prism 8.0的非线性回归曲线来计算半数效应浓度EC50的值[28-29],拟合模型为“Logistic模型”。
操作中,首先要在Excel 2010中将虫黄藻密度的数值mean和标准差SD转换为虫黄藻的逃逸率(%)和标准差率(%)作为mean(%)和SD(%),胁迫浓度值转化为log10。采用“XY”功能选项输入GraphPad Prism 8.0后选“分析工具栏(Analyses)”中的曲线拟合工具,再选择“Dose-response-Stimulation”功能选项中的“Log(agonist)vs. normalized response-Variable slope”即可拟合出曲线(图5),并自动计算出EC50的值。
由表1可知,3种珊瑚的BaP胁迫7 d的半数效应浓度(EC50)分别为指状蔷薇珊瑚47.00 μg·L−1,鹿角杯型珊瑚22.15 μg·L−1,美丽鹿角珊瑚5.38 μg·L−1。由此可知,在BaP胁迫下,3种珊瑚耐受能力依次为:指状蔷薇珊瑚>鹿角杯型珊瑚>美丽鹿角珊瑚。
表 1 3种珊瑚的半数效应浓度
Table 1. The median effective concentration of three corals
种类
Species胁迫时间/d
Exposure time回归方程
Regression equation相关系数
Correlation(R2)EC50/
(μg·L−1)95%置信区间/
(μg·L−1)
Confidence interval鹿角杯型珊瑚
Pocillopora damicornis3 Y=100/1+10^((2.912−X)× 0.642) 0.994 9 817.40 520.00~1 517.00 5 Y=100/1+10^((1.989−X) × 0.791) 0.928 8 97.47 61.95~325.90 7 Y=100/1+10^((1.345−X) × 1.377) 0.924 4 22.15 9.15~35.86 指状蔷薇珊瑚
Montipora digitata3 Y=100/1+10^((2.875−X) × 0.464) 0.912 6 750.40 402.60~1 927.00 5 Y=100/1+10^((2.017−X) × 0.868) 0.855 4 104.10 78.50-173.70 7 Y=100/1+10^((1.672−X) × 0.882) 0.903 6 47.00 37.98~59.97 美丽鹿角珊瑚
Acropora formosa3 Y=100/1+10^((1.774−X) × 1.283) 0.909 3 59.37 49.66~73.01 5 Y=100/1+10^((1.262−X) × 2.164) 0.935 6 18.27 15.29~21.39 7 Y=100/1+10^((0.730−X) × 0.988) 0.888 5 5.38 2.95~12.84 注:表中的^表示次方的运算符号。
Note: The ^ in the table represents the exponentiation symbol.
Effect of Benzo [α] pyrene Stress on the Zooxanthellae Density and Content of Chlorophyll a in Three Scleractinians
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摘要: 苯并[a]芘[Benzo(a) pyrene,BaP]是一种在环境中广泛存在的有毒的环境污染物,珊瑚是构成珊瑚礁生态系统的基石,具有重要的价值。本研究以南海3种典型的造礁石珊瑚:鹿角杯型珊瑚(Pocillopora damicornis)、指状蔷薇珊瑚(Montipora digitata)和美丽鹿角珊瑚(Acropora formosa)为研究对象,分别设置二甲基亚砜(DMSO)溶剂对照组和1.0、12.5、25.0、50.0、100.0 μg·L−1的BaP胁迫组。分别在胁迫第0天、第3天、第5天、第7天后取样。测定了珊瑚−虫黄藻共生体的共生虫黄藻密度及其叶绿素a含量的变化,并通过“Logistic模型”拟合出共生虫黄藻逃逸的半数效应浓度(EC50)。结果表明:BaP胁迫7 d的EC50分别为指状蔷薇珊瑚47.00 μg·L−1、鹿角杯型珊瑚22.15 μg·L−1、美丽鹿角珊瑚5.38 μg·L−1。综合分析各项生理指标,在BaP胁迫下,美丽鹿角珊瑚更敏感,3种珊瑚耐受力排序为:指状蔷薇珊瑚>鹿角杯型珊瑚>美丽鹿角珊瑚。Abstract: Benzo [α] pyrene (Benzo (a) pyrene, BaP) is a toxic environmental pollutant that exists widely in the environment. Corals are the cornerstones of coral reef ecosystems with important values. Three typical reef-building corals in the South China Sea, Pocillopora damicornis, Montipora digitata, Acropora formosa, and Acropora formosa, were selected and treated with BaP at the concentrations of 1 μg·L−1, 12.5 μg·L−1, 25 μg·L−1, 50 μg·L−1, 100 μg·L−1, respectively, with the DMSO solvent as control. Samples were collected on the initial 0th day; the 3rd, 5th, and 7th day after exposure to the BaP stress at different concentrations. The photosynthetic and respiratory metabolism rate, the density of the symbiotic zooxanthellae and the change of chlorophyll a content of the coral-zooxanthellae symbiont were measured, and “Logistic model” was used to fit the median effective concentration (EC50) of BaP for the symbiotic zooxanthellae. The results showed that the EC50 of BaP on the 7th day was 47.00 μg·L−1 for M. digitata, 22.15 μg·L−1 for P. damicornis, and 5.38 μg·L−1 for A. formosa. Comprehensive analysis of various physiological indicators showed that A. formosa was most sensitive to BaP among the three corals which were tolerant to BaP in the order of M. digitata > P. damicornis > A. formosa.
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Key words:
- Benzo (a) pyrene /
- stress /
- EC50 /
- sensitivity
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表 1 3种珊瑚的半数效应浓度
Table 1 The median effective concentration of three corals
种类
Species胁迫时间/d
Exposure time回归方程
Regression equation相关系数
Correlation(R2)EC50/
(μg·L−1)95%置信区间/
(μg·L−1)
Confidence interval鹿角杯型珊瑚
Pocillopora damicornis3 Y=100/1+10^((2.912−X)× 0.642) 0.994 9 817.40 520.00~1 517.00 5 Y=100/1+10^((1.989−X) × 0.791) 0.928 8 97.47 61.95~325.90 7 Y=100/1+10^((1.345−X) × 1.377) 0.924 4 22.15 9.15~35.86 指状蔷薇珊瑚
Montipora digitata3 Y=100/1+10^((2.875−X) × 0.464) 0.912 6 750.40 402.60~1 927.00 5 Y=100/1+10^((2.017−X) × 0.868) 0.855 4 104.10 78.50-173.70 7 Y=100/1+10^((1.672−X) × 0.882) 0.903 6 47.00 37.98~59.97 美丽鹿角珊瑚
Acropora formosa3 Y=100/1+10^((1.774−X) × 1.283) 0.909 3 59.37 49.66~73.01 5 Y=100/1+10^((1.262−X) × 2.164) 0.935 6 18.27 15.29~21.39 7 Y=100/1+10^((0.730−X) × 0.988) 0.888 5 5.38 2.95~12.84 注:表中的^表示次方的运算符号。
Note: The ^ in the table represents the exponentiation symbol. -
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