Collaborative Research: Microbial associations in zooplankton: significance for the marine nitrogen cycle

固氮作用是海洋生态系统中重要的氮素地球化学过程。目前的全球速率的估计表明，输入和输出的N是不平衡的，留下一个明显的不足，在N2固定在海洋N预算。然而，基于对固氮基因片段的恢复，许多潜在的固氮微生物存在于真光层及其以下，但缺乏它们对固氮的贡献的证据。本研究的目的是确定和确定潜在的重氮营养，异养原核生物占据了以前未研究的生态位在upperocean.This项目PI假设，固氮在动物浮游生物相关的微生物群落是一个重要的来源，新的N在开放的海洋，有助于海洋N预算的重要性。在贫营养、氮限制的开阔海洋沃茨中，浮游动物在其饮食中面临着同样的低氮可用性，这限制了开阔海洋中初级生产者和异养微生物的生长。 浮游动物的肠道将是一个特别适合的环境异养固氮酶的固氮酶被氧气抑制。在许多陆生昆虫中，肠道微生物的N2固定是N的来源。本研究的目的是调查是否有类似的协会发生在海洋浮游动物，生活在氮限制浮游植物。PI将调查来自N限制和非N限制海洋沃茨的桡足类与潜在或积极固定N的微生物之间的特定关联。他们还将调查这种N的命运，以研究新的N是否被纳入浮游动物宿主。主要研究目标是确定是否：1。桡足类中是否存在固氮菌群及其季节和空间变异性; 2.与桡足类相关的微生物群落正在积极地固定N2，3。固氮生物群落有助于浮游动物宿主的氮营养。 每月将在贫营养的马尾藻海收集样本，与最佳可行技术取样巡航有关。此外，夏季将在百慕大进行实验和实地研究。为了与不严重限制氮的地点进行比较，并为了开发方法，将在缅因州沿海海湾沃茨进行额外的研究。将使用克隆和测序以及逆转录酶-PCR分析所选桡足类物种及其粪便颗粒中nifH基因的存在及其表达。将采用定量PCR方法研究优势种的季节分布。厌氧培养将用于肠道微生物菌群的富集。浮游动物将与15 N2一起孵育，以通过浮游动物总体15 N含量和15 N定位的nanoSIMS测定来量化微生物N2固定活性及其N的命运。本研究的产品将包括新的微生物分离株或财团，证据有关类群的具体表现在这些微生境中的N2固定，并首次估计这一过程的重要性，为海洋N budgets.Broader impactsThis项目解决了有关海洋氮的来源的基本问题，并将测试假设关于以前被忽视的固氮生态位。由于开阔洋的生产力往往是N有限的，表征新的N源将具有全球性的影响。PI将让从K-12到研究生水平的学生参与这项研究。UMD的研究生将在研究框架内开展论文研究，几名本科生将参加UMD的实地和实验室部分。海洋微生物学课程内容将纳入马萨诸塞州新贝德福德市海洋探索中心的K-12科学课程。部分工作将作为与L.哥本哈根大学，从而加强国际联系。

Intellectual merit Nitrogen (N2) fixation is a key biogeochemical process providing new N for the marine ecosystem. Current estimates of the global rates suggest that the inputs and outputs of N are not in balance, leaving an apparent deficiency in N2 fixation in the oceanic N budget. Based on recovery of N2 fixation gene fragments, however, numerous potential N2-fixing microbes are present in the euphotic layer and below, yet evidence for their contribution to N2-fixation is lacking. The goal of this study is to identify and determine the importance of potentially diazotrophic, heterotrophic prokaryotes occupying a previously unstudied niche in the upper ocean.This project PIs hypothesize that N2 fixation in zooplankton-associated microbial communities is an important source of new N in the open ocean contributing to the oceanic N budget. In oligotrophic, N-limited open ocean waters, zooplankton are faced with the same low N availability in their diets that limits growth of primary producers and heterotrophic microbes in the open ocean. Zooplankton guts would be a particularly suitable environment for heterotrophic N2-fixers as the nitrogenase enzyme is inhibited by oxygen. In many terrestrial insects, N2 fixation by gut microbes serves as a source of N. The aim of this study is to investigate whether similar associations occur in marine zooplankton that live on N-limited phytoplankton. The PIs will investigate specific associations between copepods from N-limited and non-N limited oceanic waters with microorganisms that are potentially or actively fixing N. They will also investigate the fate of this N to study whether the new N is incorporated into the zooplankton host. The main research objectives are to determine whether:1. diazotrophic microflora exist in copepods and what is their seasonal and spatial variability, 2. microbial communities associated with copepods are actively fixing N2, 3. diazotroph communities contribute to the N nutrition of their zooplankton hosts. Monthly samples will be collected in the oligotrophic Sargasso Sea, in association with BATS sampling cruises. In addition, experiments and field studies will be conducted in Bermuda during the summer. For comparison to a site that is not severely N limited, and for methods development, additional studies will be carried out in coastal Gulf of Maine waters. Selected copepod species and their fecal pellets will be analyzed for the presence of nifH genes and their expression using cloning and sequencing and Reverse Transcriptase-PCR. Quantitative PCR will be employed to investigate the seasonal distributions of dominant phylotypes. Anaerobic cultivation will be used for the enrichment of the gut microflora. Zooplankton will be incubated with 15N2 to quantify microbial N2 fixation activity and its fate of N through zooplankton overall 15N content and nanoSIMS determination of 15N localization. Products of this study will include novel microbial isolates or consortia, evidence regarding taxa-specific expression of N2 fixation in these microhabitats, and a first estimate for the importance of this process for the oceanic N budget.Broader impactsThis project addresses fundamental issues regarding the sources of oceanic nitrogen and will test hypotheses regarding previously overlooked diazotrophic niches. As open ocean productivity is frequently N limited, characterization of new sources of N will have global impacts. The PIs will engage students from K-12 to graduate levels in this research. Graduate student at the UMD will develop their dissertation research within the framework of the study, and several undergraduate students will participate in field and lab components at UMD. Marine microbiology course content will be built into the K-12 science curriculum of the Ocean Explorium of the city of New Bedford, Massachusetts. Part of the work will be performed as a collaborative project with L. Riemann, Univ. of Copenhagen, thus strengthening international connections.