Ecology of eukaryote microbes in the deep North Atlantic

在微生物领域，生命的三个领域之一--真核生物--在深海研究中几乎没有受到关注。这与以下事实形成鲜明对比：在所有已知的水生环境中，原核生物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，并以转移的物质和能量的数量衡量。就体积而言，深海是最大的生物群，尽管它在长期的生物地球化学循环中发挥了巨大作用，但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，也不是同质的。最近的数据表明，暗海洋中的生物活动(如呼吸速率、细菌二次生产和各种其他指标)远远高于所有已知有机碳燃料来源(即POC通量、DOC对流、就地生产和浮游动物的主动运输)的预期。深海水团代表着高度多样化的生物地理区域，具有不同的群落和颗粒分布。此外，由于深海微生物表现出的摄食阈值、寒冷温度、极端压力和独特的适应能力，生物活动规则不能简单地从实验室培养和对表层微生物的实验中推断出来。这项研究的重点是真核微生物群落在深海生态中的基础作用，最重要的假设是原生生物是可利用有机碳的敏感生物学指标。我们有充分的理由相信，与颗粒清单、沉积物捕捉器、同位素比率或基于地表产量和理论通量衰减的模型相比，微生物真核生物及其活动是有机碳“新”来源的更好指标。然而，为了使这些新的生物指示器发挥作用，人们需要将活细胞与垂死和死亡的细胞分开，将食菌者与腐生菌分开，将不活跃的休眠阶段与活跃的原核生物分开，将营养和取食阶段的配子和游动孢子分开，将位于颗粒上的那些与自由悬浮在水柱中的分开。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。学术价值：这项研究确定了北大西洋深处大片地理区域内真核微生物的生态作用。这项研究采用了两种完全不同的实验设计，它们利用不同的时间尺度：1)呼吸活动和细菌的短期孵化(~72小时)，结合从研究船上进行的大范围地理区域的高分辨率采样；以及2)长期孵化(=/4周)，使用自由落体(非系泊)运载工具测量下沉颗粒的定植和真核微生物的生长，这是首次尝试直接在深海中进行生理速率测量。方法包括新的细菌示踪剂，单细胞呼吸培养，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微米的分辨率捕捉最大深度6000米的图像。更广泛的影响：该项目支持本科生和研究生研究，并加强国际海洋学研究所举办的生物海洋学课程的本科生课程。将在波多黎各为任职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被安装在汉普顿路的一个主要博物馆和解释中心。这项研究对开发海洋研究的新技术作出了重大贡献，重点是深海环境，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.