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

氮（N2）固定是为海洋生态系统提供新氮的关键生物地球化学过程。目前对全球速率的估计表明，氮的输入和输出并不平衡，在海洋氮收支中留下了明显的氮固定不足。然而，根据固氮基因片段的恢复，在光层及以下存在许多潜在的固氮微生物，但缺乏证据证明它们对固氮的贡献。本研究的目的是确定和确定潜在重氮营养的重要性，异养原核生物占据了以前未研究过的上层海洋生态位。本项目pi假设浮游动物相关微生物群落的氮固定是开放海洋中新氮的重要来源，有助于海洋氮收支。在低营养、氮限制的开放海洋水域，浮游动物面临着同样低的饮食氮利用率，这限制了开放海洋中初级生产者和异养微生物的生长。由于氮酶被氧气抑制，浮游动物的肠道将是一个特别适合异养固氮生物的环境。在许多陆生昆虫中，肠道微生物对N2的固定是n的来源，本研究的目的是调查以n限制浮游植物为食的海洋浮游动物是否也存在类似的关联。pi将研究来自N限制和非N限制海洋水域的桡足类动物与潜在或主动固定N的微生物之间的特定关系。他们还将研究这些N的命运，以研究新N是否被纳入浮游动物宿主。主要研究目的是确定是否：1。桡足类重氮营养菌群的存在及其季节和空间变异性；桡足类相关微生物群落积极固定N2, 3。重氮营养群落对其浮游动物宿主的氮营养有贡献。每月将在低营养马尾藻海收集样本，并配合bats采样巡航。此外，夏季将在百慕大进行实验和实地研究。为了与不受严重氮限制的地点进行比较和开发方法，将在缅因湾沿岸水域进行额外的研究。选择的桡足动物及其粪便颗粒将通过克隆、测序和逆转录- pcr分析nifH基因的存在及其表达。将采用定量PCR方法研究优势种型的季节分布。厌氧培养将用于肠道菌群的富集。用15N2孵育浮游动物，通过浮游动物总15N含量和纳米sims测定15N定位来量化微生物固氮活性及其对N的归宿。这项研究的成果将包括新的微生物分离物或群落，关于这些微栖息地中N2固定的分类特异性表达的证据，以及对这一过程对海洋N收支重要性的首次估计。更广泛的影响该项目解决了关于海洋氮来源的基本问题，并将测试关于以前被忽视的重氮营养生态位的假设。由于开放海洋的生产力经常受到氮的限制，对氮新来源的描述将产生全球性影响。pi将让从K-12到研究生阶段的学生参与这项研究。UMD的研究生将在研究框架内进行论文研究，一些本科生将参与UMD的现场和实验室组成部分。海洋微生物学课程内容将被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。部分工作将作为与哥本哈根大学L. Riemann的合作项目进行，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.