Ecology of eukaryote microbes in the deep North Atlantic

在微生物领域，三大生命领域之一-真核生物-在深海研究中很少受到注意。这与以下事实形成鲜明对比：在所有已知的水生环境中，通过物质和能量转移的量来衡量，原核细胞和真核细胞之间的联系是地球上最重要的营养相互作用之一。就体积而言，深海是最大的生物群落，尽管它在长期的地球化学循环中发挥着巨大作用，但它在很大程度上被忽视了。深海的生物活动在空间和时间上既不是微不足道的，也不是单一的。最近的数据表明，黑暗海洋中的生物活动（如呼吸率，细菌二次生产和各种其他指标所证明的）远远高于所有已知有机碳燃料来源的预期（即，POC通量，DOC对流，原位生产和浮游动物的主动运输）。深海中的水团代表了具有不同群落和颗粒分布的高度多样的海洋地理区域。此外，由于深海微生物的摄食阈值、低温、极端压力和独特的适应性，生物活动规则不能简单地从实验室培养和对表面微生物的实验中推断出来。本研究的重点是真核微生物群落在深海生态中的基本作用，其总体假设是原生生物代表可利用有机碳的敏感生物指标。有充分的理由相信，微生物真核生物和它们的活动是更好的指示“新”的有机碳来源比颗粒库存，沉积物陷阱，同位素比，或基于表面生产和理论通量衰减模型。然而，为了使这些新的生物指示剂发挥作用，需要将活细胞与垂死和死亡细胞、食菌动物与腐养生物、不活跃的休眠阶段与活跃地以原核生物为食的阶段、配子和游动孢子与营养阶段和进食阶段、以及位于颗粒上的配子和游动孢子与自由悬浮在水柱中的配子和游动孢子分开。这些组中的每一组代表每个电池的能量和碳需求的不同水平。智力价值：这项研究确定了北大西洋深处真核微生物在大地理区域的生态作用。该研究结合了两种根本不同的实验设计，利用不同的时间尺度：（1）短期孵化（约72小时）的呼吸活动和噬菌性，结合从研究船上对大地理区域的丰度进行的高分辨率采样，（2）长期孵化（=/ 4周）使用自由落体法测量沉降颗粒的定殖和真核微生物的生长（无系留）潜水器代表直接在深海中进行生理速率测量的首次尝试。方法包括新的噬菌体示踪剂、单细胞呼吸孵育、利用荧光原位杂交进行分类鉴定、单细胞基因组学以及第一台深海全息显微镜以5微米分辨率捕捉最大深度为6 000米的图像。更广泛的影响：该项目支持本科生和研究生的研究，并加强由PI举办的生物海洋学课程的本科课程。将在波多黎各为代表人数不足的群体举办讲习班。一个关于微生物在海洋中的作用的永久展览将安装在汉普顿路的一个主要博物馆和解释中心。这项研究大大有助于开发以深海环境为重点的海洋学研究新技术，并支持国际科学研究合作。

In the microbial realm, one of the three domains of life -- the Eukarya -- has received little attention in deep-sea research. This stands in contrast to the fact that in all known aquatic environments, and measured by the amount of material and energy transferred, the link between prokaryotic and eukaryotic cells is one of the most significant trophic interactions on Earth. In terms of volume, the deep sea is the largest biome, and despite its tremendous role in long-term biogeochemical cycles, it has largely been neglected. Biological activity in the deep sea is neither negligible nor homogeneous in space and time. Recent data suggest that biological activity in the dark ocean (as evidenced by respiration rates, bacterial secondary production and a variety of other metrics) is much higher than anticipated from all known organic carbon fuel sources combined (i.e., POC flux, DOC convection, in situ production and active transport by zooplankton). Water masses in the deep ocean represent highly-diverse biogeographic regions with distinct communities and particle distributions. Moreover, because of feeding thresholds, cold temperatures, extreme pressures and unique adaptations that deep-sea microbes exhibit, biological activity rules cannot simply be extrapolated from laboratory cultures and from experiments with surface-dwelling microbes. This study focuses on the fundamental role of eukaryotic microbial communities in deep-sea ecology with the overarching hypothesis that protists represent sensitive biological indicators of utilizable organic carbon. There is good reason to believe that microbial eukaryotes and their activities are better indicators of "new" sources of organic carbon than particle inventories, sediment traps, isotope ratios, or models based on surface production and theoretical flux attenuation. For these new biological indicators to work, however, one needs to separate live from the moribund and dead cells, the bacterivores from saprotrophs, the inactive resting stages from those actively feeding on prokaryotes, the gametes and zoospores from vegetative and feeding stages, and those located on particles from the ones freely suspended in the water column. Each of these groups represents different levels of per-cell energy and carbon requirements. Intellectual merit: This study determines the ecological role of eukaryotic microbes in the deep North Atlantic over large geographic regions. The research incorporates two fundamentally different experimental designs that capitalize on different time scales: 1) Short-term incubations (~72 hours) of respiratory activity and bacterivory combined with a high resolution sampling of abundances across large geographic regions performed from a research vessel, and 2) Long-term incubations (=/ 4 weeks) measuring colonization of sinking particles and growth of eukaryotic microbes using free-falling (untethered) vehicles representing the first attempt of physiological rate measurements directly in the deep sea. Methods include new tracers for bacterivory, incubations for single-cell respiration, taxonomic identification using fluorescence in situ hybridization, single-cell genomics, and the first of its kind deep-sea holographic microscope capturing images to a maximum depth of 6000 m at 5 micrometer resolution. Broader Impacts: This project supports undergraduate and graduate research and enhances undergraduate curriculum in biological oceanography courses held by the PI. Workshops for underrepresented groups will be held in Puerto Rico. A permanent exhibit on the role of microbes in the ocean will be installed at a major museum and interpretation center in Hampton Roads. This research contributes significantly to the development of new technology for oceanographic research with emphasis on deep-sea environments and supports international scientific research collaborations.