Pickled Protists or Community Uniquely Adapted to Hypersalinity

原生生物是微生物食物网的重要组成部分，在全球生物地球化学循环中发挥核心作用，因此是维持任何生态系统健康运作的关键角色。在过去的几年里，在一系列极端环境中发现了丰富多样的原生生物，这表明真核生物生命的前沿仍在探索中。就在最近，在地中海东部约3500米深处发现了科学界已知的最极端的海洋环境之一，即深层高盐缺氧盆地(DHAB)。这些盆地的特点是盐分浓度极高(达到饱和)，这被认为是生命的诅咒。相反，这些盆地的水中存在着高度多样化的细菌群落。除了对这一提议的初步研究表明盐跃层沿线和盐跃层以下的水柱中有多样化和活跃的原生生物组合外，这些DHAB在很大程度上仍然没有被探索到真核生物的生命形式。DHAB的沉积物根本没有勘探过原生生物。调查人员将在一次短暂的巡航中收集水柱和沉积物样本，前往两个具有不同盐水化学成分的盆地。将采用令人兴奋的分子、独立于栽培和以培养为基础的方法相结合的方法来研究Bannock盆地和Discovery盆地的微生物群落，并确定海洋原生生物群落对高盐度、缺氧环境的适应策略及其由于捕食活动对生物地球化学循环的潜在影响的程度、主要原生生物保持细菌或古生物共生体的程度以及这些共生体的特征。可采用的方法包括基于18S rDNA基因的基于RNA的多样性序列分析，群落组成和系统类型丰富度的统计分析，使用经典和微电极方法的水柱和沉积物的地球化学记录，使用mRNAs的3‘-UTR片段的表达谱分析，似乎赋予高盐度适应的蛋白质的完整基因转录本的测序，蛋白质组特征分析，FISH-SEM来表征新型极端嗜盐菌，CARD-FISH来鉴定真核动物猎物和可能的共生体，以及TEM来评估常见海底形态和内离子的存在。例如，考虑到地球上某些地区气温上升和干旱的预测，高盐度栖息地的数量可能会急剧增加，导致这个生态系统在全球范围内变得更加重要。因此，了解这些生境中的生态系统将有助于预测由于全球变化而产生的未来生态系统功能。从不同的角度来看，揭示适应高盐度的机制已成为生物科学和与这些适应相关的自然产品的潜在商业开发的一个主要目标。此外，该项目还包括从中学到研究生学习的各种教育内容，以及为中学科学教师和普通公众提供的外展机会。例如，这个项目将部分资助一名研究生，他将加入研究游轮，获得第一手海洋学经验，并增加他们对海洋微生物生态和生理学的知识。这一项目的方法和成果将在为来自世界各地的中学和中学科学教师举办的一个著名的年度讲习班上介绍。

Protists are an essential component of microbial food webs and play a central role in global biogeochemical cycles, and thus are key players in sustaining the healthy functioning of any ecosystem. Over the past few years a rich diversity of protists has been revealed in a range of extreme environments, indicating that the frontiers of eukaryotic life are still being explored. Only recently, one of the most extreme marine environments known to science was discovered in the eastern Mediterranean Sea at a depth of ~3500m, namely deep hypersaline anoxic basins (DHABs). These basins are characterized by extremely high salt concentrations (up to saturation) that have been considered anathema to life. Instead, highly diverse communities of bacteria exist in the waters of these basins. With the exception of a preliminary study to this proposal that indicated a diverse and active assemblage of protists in the water column along the halocline and below the halocline, these DHABs remain largely unexplored regarding eukaryotic life forms. The sediments of the DHABs have not been explored for protists at all. The investigators will collect water column and sediment samples on a short cruise to two basins with different brine chemistries. An exciting combination of molecular, cultivation-independent and culture-based approaches will be used to study the microbial communities of Bannock and Discovery Basins as well as determine adaptive strategies of marine protist communities to hypersaline, anoxic environments and the degree of their potential impact on biogeochemical cycling as a result of their predation activities, the degree to which the dominant protists maintain bacterial or archaeal symbionts, and the identity of those symbionts. Methods to be employed include RNA-based sequence analysis of diversity based on 18S rDNA genes, statistical analyses of community composition and phylotype richness, geochemical documentation of the water column and sediments using classical and microelectrode approaches, expression profiling using 3'-UTR fragments of mRNAs, sequencing of complete gene transcripts for proteins appearing to confer adaptation to hypersalinity, analysis of the proteome signatures, FISH-SEM to characterize novel extremophiles, CARD-FISH to identify eukaryote prey and putative symbionts, and TEM to assess morphology and endobiont presence in common benthic morphotypes.Broader Impacts.Hypersaline environments rank highly in the list of extreme systems that have attracted increasing notice in science as well as by the lay public. For example, considering predictions of increasing temperatures and drought in certain regions of our planet, the number of hypersaline habitats may increase dramatically causing this ecosystem to gain importance on a global scale. Thus, an understanding of the ecosystem in these habitats will help predict future ecosystem functioning due to global change. From a different perspective, revealing the mechanisms of adaptation to high salinity has become a major objective, both for biological science and for potential commercial exploitation of natural products associated with those adaptations. Additionally, this project has varied educational components, ranging from middle school to graduate studies, and outreach opportunities for secondary school science teachers and the lay public. For example, this project will partially support one graduate student, who will join the research cruise to gain first-hand oceanographic experience and augment their knowledge of marine microbial ecology and physiology. The methodologies and results of this project will be presented at a well-established annual workshop for middle and secondary science teachers from around the world.