Resolving the trophic connection between protistan grazers and mesozooplankton in marine food webs using amino acid-specific stable isotope analyses

智力优势：根据初级产品消费量的全球平均测量，海洋食物网中较低营养水平的原生生物消费者的能量耗散和元素循环具有足够的规模，可以强烈改变向较高水平消费者转移的物质效率并出口。我们目前对这些微生物食物网的步骤知之甚少，也不知道它们在区域或时间上是如何变化的，也不知道它们是如何被气候变化所改变的。氨基酸的复合同位素分析（CSIA-AA）为我们理解微生物食物网结构和营养通量提供了一种方法，该方法基于中浮游动物作为直接取食浮游植物和通过原生微浮游动物间接转移的通量的时间积分器的营养位置（TP）。为验证这一想法而进行的初步实验室实验表明，该方法的标准应用，即使用标记苯丙氨酸作为初级生产者基线的代表性来源AA，并将标记谷氨酸作为营养富集的指示物AA，不会对原菌食草动物产生可测量的营养步骤信号。然而，结果也表明，另一种高周转率的AA，丙氨酸，在原生动物和后生动物的消费者中富集，导致现场收集的标本中浮游动物的总磷估计比基于标记谷氨酸的估计高得多。本研究项目将检验标记丙氨酸为海洋食物网中原生生物步骤的营养富集提供可量化和一致的指标的假设。这项研究将涉及三个主要因素。首先，将在实验室进行控制实验，比较以浮游植物为食的具有代表性的纤毛虫和鞭毛虫食草动物对丙氨酸和其他氨基酸的15N富集程度，并评估浮游植物通过原生食草动物到悬浮进食桡足动物的两步富集。其次，利用CSIA-AA对太平洋四个不同生态区野外采集的中浮游动物进行分析，以测试通过后生动物营养步骤（将悬浮食性物种与主要食肉动物进行比较）富集丙氨酸的转移，并评估TP指数在广泛生态条件下随营养结构的差异。最后，将CSIA-AA对大小结构浮游动物TP的评估整合到四个区域（赤道太平洋、亚热带北太平洋、加利福尼亚海流和哥斯达黎加巨丘）的氮流反演模型中，作为解决和比较不同生态条件下微生物食物网通量的主要约束条件。对中浮游动物营养地位进行适当校准的CSIA-AA评估，将为较低层食物网结构的区域间比较、气候变化的时空趋势评估、海洋生态系统模型验证以及更好地理解海洋渔业的较低层食物网能量约束提供新的有价值的方法。更广泛的影响：该项目将涉及各级（博士后、研究生、本科生和K-12）的指导关系，重点是促进海洋生态学和海洋学领域未被充分代表的年轻女性的科学兴趣和职业道路。本科生研究人员将参与相关的实践研究活动，作为斯克里普斯本科生研究奖学金（SURF）和CCE-LTER夏季REU项目的一部分。在K-12阶段，调查人员将与海洋发现研究所（Ocean Discovery Institute）和圣地亚哥拓展你的视野（Expanding Your Horizons San Diego）合作，前者为圣地亚哥城市青年提供科学和教育，后者为来自少数族裔的女孩提供科学活动。这项研究的结果将有助于理解构建食物网基础的机制，它的可变性和弹性。这些结果将对全球生物地球化学循环和海洋渔业模型具有重要意义。

Intellectual merit: Energy dissipation and elemental cycling by protistan consumers in lower trophic levels of ocean food webs are of sufficient magnitude, based on global mean measures of the amount of primary production consumed, to strongly alter the efficiencies of material transfers to higher-level consumers and to export. We presently know very little about these microbial food web steps, how they vary regionally or temporally, or how they might be altered by climate change. Compound Specific Isotope Analysis of Amino Acids (CSIA-AA) offers an approach for advancing our understanding of microbial food web structure and trophic fluxes based on the trophic positions (TP) of mesozooplankton as temporal integrators of the fluxes from direct feeding on phytoplankton and indirect transfers via protistan microzooplankton. Preliminary laboratory experiments to test this idea have demonstrated that the standard application of the method, using labeled phenylalanine as the representative source AA for the primary producer baseline and labeled glutamic acid as the indicator AA for trophic enrichment, does not produce a measureable trophic-step signal for protistan grazers. However, the results have also shown that an alternative high-turnover AA, alanine, strongly enriches in protistan as well as metazoan consumers, and leads to substantially higher TP estimates of mesozooplankton in field-collected specimens than that based on labeled glutamic acid. This research project will test the hypothesis that labeled alanine provides a quantifiable and consistent index of trophic enrichment for protistan steps in marine food webs. The research will involve three major elements. First, controlled laboratory experiments will be conducted with chemostat systems to compare 15N enrichments of alanine to other AAs for a representative suite of ciliate and flagellate grazers feeding on phytoplankton, and to evaluate the two-step enrichment from phytoplankton via a protistan grazer to a suspension-feeding copepod. Second, field-collected mesozooplankton from four distinct ecological regions of the Pacific Ocean will be analyzed by CSIA-AA to test the transfer of alanine enrichment through a metazoan trophic step (comparing suspension feeding species to primary carnivores) and to assess how the TP index differs with trophic structure over a broad range of ecological conditions. Last, CSIA-AA assessments of TP for size-structured zooplankton will be integrated into inverse models of nitrogen flows in the four regions (equatorial Pacific, subtropical North Pacific, California Current and Costa Rica Dome) as a major constraint for resolving and comparing fluxes through the microbial food web over the range of ecological conditions. A properly calibrated CSIA-AA assessment of mesozooplankton trophic position will provide a new and valuable approach for regional intercomparisons of lower-level food web structure, for assessing temporal and spatial trends in climate change, for ocean ecosystem model validation, and for better understanding of lower food-web energetic constraints on ocean fisheries. Broader Impacts: This project will involve mentoring relationships at all levels (postdoctoral, graduate, undergraduate and K-12) with an emphasis on advancing the scientific interests and career paths of young underrepresented women in marine ecology and oceanography. Undergraduate researchers will be engaged in hands-on relevant research activities as part of the Scripps Undergraduate Research Fellowship (SURF) and CCE-LTER summer REU programs. At the K-12 level, the investigators will work with the Ocean Discovery Institute, which brings science and education to San Diego urban youth, and Expanding Your Horizons San Diego, a program that provides scientific activities to girls from underrepresented minorities. Results from the study will contribute to understanding the mechanisms structuring the base of the food web, its variability and resilience. The results will have important implications for models of global biogeochemical cycling and ocean fisheries.