Detecting in situ copepod diet diversity using molecular technique: development of a copepod/symbiotic ciliate-excluding eukaryote-inclusive PCR protocol.
Detecting in situ copepod diet diversity using molecular technique: development of a copepod/symbiotic ciliate-excluding eukaryote-inclusive PCR protocol.
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使用分子技术原位检测桡足类饮食多样性：开发桡足类/共生纤毛虫排除真核生物的 PCR 方案
DOI：
10.1371/journal.pone.0103528
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Lin S
中科院分区：
文献类型：
--
作者：
Hu S;Guo Z;Li T;Carpenter EJ;Liu S;Lin S
在原地桡足类的饮食多样性的知识是至关重要的，准确地描述远洋食物网结构，但由于缺乏一个容易适用的方法是具有挑战性的。为了使分析与整个桡足类衍生的DNA，我们开发了一个桡足类排除18 S rDNA为基础的PCR协议。虽然它是有效的抑制扩增桡足类18 S rDNA，它的适用性，以检测不同的真核生物在单一和混合物种尚未得到证明。此外，该协议遭受的问题，共生纤毛虫的序列在检索的18 S rDNA文库中的过度代表。在这项研究中，我们设计了一个封闭引物，使组合引物集（桡足类/共生纤毛虫排除真核生物常见：CEEC）抑制共生纤毛虫序列的PCR扩增，同时最大限度地扩大真核生物扩增的范围。我们首先通过PCR扩增来自16种桡足类、37种桡足类潜在猎物的代表性生物和天然浮游生物样品的DNA来检验CEEC的特异性和有效性，然后通过检测实验室饲养和现场收集的桡足类的食物来评估CEEC重建食物组成的效率。结果表明，CEEC引物组能成功扩增出大范围分离物种和混合物种的18 SrDNA，而对桡足类和目标共生纤毛虫的18 SrDNA扩增则表现出抑制作用，表明CEEC在桡足类猎物特异性检测中具有普遍性。成功地检索了实验室中提供给桡足类的所有预定食物，这表明基于CEEC的方案可以准确地重建桡足类的饮食，而不会干扰DNA样品中存在的桡足类及其相关的纤毛虫。我们最初的应用程序，以分析现场收集的桡足类的食物组成发现不同的猎物物种，包括目前已知的，和那些不被怀疑，桡足类猎物。虽然需要进行测试，但该方案为描绘桡足类的原位膳食组成提供了有用的策略。
Knowledge of in situ copepod diet diversity is crucial for accurately describing pelagic food web structure but is challenging to achieve due to lack of an easily applicable methodology. To enable analysis with whole copepod-derived DNAs, we developed a copepod-excluding 18S rDNA-based PCR protocol. Although it is effective in depressing amplification of copepod 18S rDNA, its applicability to detect diverse eukaryotes in both mono- and mixed-species has not been demonstrated. Besides, the protocol suffers from the problem that sequences from symbiotic ciliates are overrepresented in the retrieved 18S rDNA libraries. In this study, we designed a blocking primer to make a combined primer set (copepod/symbiotic ciliate-excluding eukaryote-common: CEEC) to depress PCR amplification of symbiotic ciliate sequences while maximizing the range of eukaryotes amplified. We firstly examined the specificity and efficacy of CEEC by PCR-amplifying DNAs from 16 copepod species, 37 representative organisms that are potential prey of copepods and a natural microplankton sample, and then evaluated the efficiency in reconstructing diet composition by detecting the food of both lab-reared and field-collected copepods. Our results showed that the CEEC primer set can successfully amplify 18S rDNA from a wide range of isolated species and mixed-species samples while depressing amplification of that from copepod and targeted symbiotic ciliate, indicating the universality of CEEC in specifically detecting prey of copepods. All the predetermined food offered to copepods in the laboratory were successfully retrieved, suggesting that the CEEC-based protocol can accurately reconstruct the diets of copepods without interference of copepods and their associated ciliates present in the DNA samples. Our initial application to analyzing the food composition of field-collected copepods uncovered diverse prey species, including those currently known, and those that are unsuspected, as copepod prey. While testing is required, this protocol provides a useful strategy for depicting in situ dietary composition of copepods.