热带海洋学报

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长棘海星(Acanthaster planci)幼体特异性PCR检测技术与应用

张颖1,杨栎潼2,刘冰1, 3,郑凡昱1,罗鹏1,陈偿1, 4   

  1. 1. 中国科学院南海海洋研究所,中国科学院热带海洋生物资源与重点实验室/广东省应用海洋生物学重点实验室,广东 广州 510301;

    2. 厦门大学马来西亚分校,中国东盟海洋技术学院,雪兰莪,雪邦 43900

    3. 中国科学院大学,北京 10049;

    4. 中国科学院南海海洋研究所,海南西沙海洋环境国家野外科学观测研究站,广东 广州 510301

  • 收稿日期:2022-01-20 修回日期:2022-05-09 出版日期:2022-05-19 发布日期:2022-05-19
  • 通讯作者: 陈偿
  • 基金资助:
    财政部和农业农村部:国家现代农业产业技术体系资助

Specific PCR detection for Acanthaster planci larvae and its application

ZHANG Ying1, YANG Litong2, LIU Bing1,3, ZHENG Fanyu1, LUO Peng1, CHEN Chang1, 4   

  1. 1. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, Guangdong 510301, China

    2. China-ASEAN College of Marine Sciences, Xiamen University Malaysia, Sepang, Selangor Darul Ehsan, 43900, Malaysia

    3. University of Chinese Academy of Sciences, Beijing 100049, China

    4. Xisha Marine Environment National Observation and Research Station, Hainan, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, Guangdong 510301, China

  • Received:2022-01-20 Revised:2022-05-09 Online:2022-05-19 Published:2022-05-19
  • Supported by:
    China Agriculture Research System of MOF and MARA

摘要: 长棘海星(Acanthaster planci)的暴发是导致我国南海乃至印度-太平洋海域珊瑚礁退化的主要原因之一。浮游幼体的密度是决定成体种群是否暴发的重要指标,但是由于幼体肉眼不可见且不易分辨,常规调查和显微镜观察均无法有效检测到自然海域的长棘海星幼体,因此迫切需要开发一种高灵敏性且特异性的长棘海星幼体检测技术。本研究针对长棘海星幼体线粒体细胞色素氧化酶亚基I(mtCOI,mitochondrial cytochrome oxidase subunit I)基因序列,建立了基于聚合酶链式反应(PCR,polymerase chain reaction)的长棘海星幼体特异性检测技术,并对西沙七连屿珊瑚礁海域的长棘海星幼体进行了检测。结果表明,设计筛选的4对特异性引物均可以扩增长棘海星ApmtCOI基因片段,且与蓝指海星、面包海星、粒皮海星和吕宋棘海星没有交叉反应。而且我们发现,在退火温度为58.5℃时,引物2aooniF/2aooniR的特异性最佳,具有较高的灵敏度,可检测到皮克级的长棘海星基因组DNA。我们利用该技术检测了西沙七连屿海域长棘海星幼体分布情况,发现10月底西沙七连屿珊瑚礁海域能检测到长棘海星幼体,且幼体分布并不均匀。因此,该检测技术可作为今后长棘海星幼体种群监测的有效方法。

关键词: 长棘海星, 细胞色素氧化酶亚基I, 聚合酶链式反应, 幼体

Abstract: The outbreak of Crown-of -Thorns Seastar (CoTS, Acanthaster planci) is one of the main causes to coral reef degradation in the South China Sea and the Indo-Pacific region. The density of CoTS larvae is an important indicator to determine whether the outbreak of CoTS adult population occurred or not. However, as the larvae are not visible and difficult to be distinguished, conventional investigation and microscope observation cannot effectively distinguish the CoTS larvae in natural seawaters. Therefore, it is needed to develop a sensitive and specific method for detecting CoTS larvae. This study established specific polymerase chain reaction (PCR) method to detect CoTS larvae based on CoTS mitochondrial cytochrome oxidase subunit I gene (ApmtCOI), and this method was applied to detect the CoTS larvae in the coral reef around Qilianyu Island, Xisha. The results showed that the designed and screened four pairs of specific primers could succeed in amplifying ApmtCOI gene, and had no cross-reaction with Linckia laevigata, Culcita novaeguineae, Choriaster granulatus and Echinaster luzonicus. Moreover, the primer 2aooniF/2anooiR had the best specificity and higher sensitivity when annealing temperature was 58.5°C, which could detect the CoTS genomic DNA in pictogram grade. Furthermore, this method had succeeded in detecting the CoTS larvae in Qilianyu Island of Xisha at the end of October, and we found that the distribution of CoTS larvae was not uniform. Therefore, this detection technology can be used as an effective method for monitoring CoTS larvae population in the future.

Key words: Acanthaster planci, Cytochrome oxidase subunit I, Polymerase Chain Reaction, Larvae