Quantitative PCR to estimate copepod feeding
Quantitative PCR to estimate copepod feeding
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DOI：
10.1007/s00227-007-0830-x
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发表时间：
2008-02-01
期刊：
影响因子：
2.4
通讯作者：
Artigas, L. Felipe
中科院分区：
文献类型：
--
作者：
Nejstgaard, Jens C.;Frischer, Marc E.;Artigas, L. Felipe
桡足类动物作为浮游生物的食草动物和许多捕食者的主要猎物，在海洋食物网中扮演着核心角色。因此，量化它们特定的营养相互作用对于理解桡足类动物在海洋过程中的作用至关重要。然而，由于方法上的限制，在不依赖于费力的侵入性和潜在偏见的孵化方法的情况下，仍然很难对原地桡足动物的摄食进行调查。基于pcr方法的最新进展已经证明了根据猎物DNA序列直接识别桡足动物饮食的可行性。然而，从这些方法中获得定量信息仍然具有挑战性。本研究系统地建立了一种针对18S rRNA基因片段的定量PCR （qPCR）方法，以估计特定种类捕食藻类的桡足类肠道含量。这些结果首先与对照实验室研究中基于荧光的桡足动物Calanus finmarchicus肠道含量估计进行了比较。在随后的实地研究中，我们比较了显微镜和qPCR获得的长角田鼠（Temora longicornis）和克氏阿卡蒂亚（Acartia clausi）在自然繁殖中取食球状褐藻（Phaeocystis globosa）的取食率。这些研究表明，通过qPCR、肠道色素和直接显微镜得出的肠道含量估计值之间存在半定量关系。然而，基于qPCR方法的肠道含量的绝对估计值始终低于预期。这似乎不能用所使用的提取方法来解释，也不能用PCR反应中非目标（捕食者）DNA的干扰来解释，而是说明了被捕食者特异性核酸的消化。此外，浮游植物的18S rDNA靶基因拷贝数随生长阶段而变化。然而，当猎物在环境水中的靶基因拷贝数被量化时，qpcr方法可以与其他方法进行比较，然后用于半定量地估计桡足类对特定猎物的相对放牧，而不涉及进一步的孵化。与肠道荧光和直接显微观察相比，基于dna的分子方法的一个明显优势是能够检测非色素和浸泡过的猎物。未来的研究应致力于纠正猎物DNA的破坏，并对其他方法进行广泛的校准，以实现就地摄食率的定量测量。
Copepods play a central role in marine food webs as grazers of plankton and as key prey for many predators. Therefore, quantifying their specific trophic interactions is critical for understanding the role of copepods in ocean processes. However, because of methodological constraints, it remains difficult to investigate in situ copepod feeding without reliance on laborious intrusive and potentially biased incubation approaches. Recent advances in PCR-based methodologies have demonstrated the feasibility of directly identifying copepod diets based on prey DNA sequences. Yet, obtaining quantitative information from these approaches remains challenging. This study presents results of systematic efforts to develop a quantitative PCR (qPCR) assay targeted to 18S rRNA gene fragments to estimate copepod gut content of specific species of prey algae. These results were first compared to gut content estimates based on fluorescence in the copepod Calanus finmarchicus fed monocultures of two different microalgae species in controlled laboratory studies. In subsequent field studies, we compared feeding rates obtained by microscopy and qPCR for Temora longicornis and Acartia clausi feeding on the haptophyte Phaeocystis globosa in natural blooms. These investigations demonstrate a semi-quantitative relationship between gut content estimates derived from qPCR, gut pigment, and direct microscopy. However, absolute estimates of gut content based on qPCR methodology were consistently lower than expected. This did not appear to be explained by the extraction methods used, or interference by non-target (predator) DNA in the PCR reactions, instead suggesting digestion of prey-specific nucleic acids. Furthermore, the 18S rDNA target gene copy number of the phytoplankton varied with growth phase. Nonetheless, when prey target gene copy number in the ambient water is quantified, the qPCR-approach can be compared to other methods, and then used to semi-quantitatively estimate relative copepod grazing on specific prey in situ without involving further incubations. A distinct advantage of a DNA-based molecular approach compared to gut fluorescence and direct microscopic observation, is the ability to detect non-pigmented and macerated prey. Future studies should aim to correct for breakdown in prey DNA and perform extensive calibrations to other methods in order to achieve a quantitative measure of feeding rates in situ.