Invasion of the Bodysnatchers? Investigations into the Physiological State of DHAB Metazoans

智力价值：虽然人们几十年前就知道后生动物周期性地、暂时地或半永久性地生活在缺氧的生境中，但还没有人被证明在没有氧气的情况下完成了整个生命周期。最近的一项引人注目的观察表明，在没有溶解氧气的情况下，后生氯藻完成了完整的生命周期，这在生理学和进化领域引发了问题。收集厌氧动物的栖息地是地中海东部3公里水深的深度高盐度缺氧卤水(DHAB)的沉积物。DHAB是科学界已知的最极端的海洋环境之一，水化学被认为是真核生物所憎恶的。虽然厌氧后生动物的可能性令人兴奋，但在重写生物教科书之前，还有其他潜在的解释值得研究。另一种情况是，残留的后生动物体内居住着厌氧细菌和/或古生菌。该项目的总体目标是确定三种DHAB中的优势类群是否代表活的种群。由于残留的DNA可以在缺氧环境中长时间保存，该项目将包括用于RNA分析的原位保存。此外，由于在这些缺氧的沉积环境中也有一些保存RNA的机会，因此这项研究将包括对更短暂的mRNA的分析，以及透射电子显微镜(TEM)的分析。在资助的邮轮上增加三个船日，以采样用于其他目的的DHAB，ROV将用于就地保存样品。具体目的是：(1)利用RNA和DNA分析来确定在每个DHAB的两个优势后生动物分类群中，后生动物核糖体RNA和功能基因在原位保存时是否活跃。(2)利用RNA分析和FISH/CARD FISH鉴定与DHAB后生动物相关的原核生物。(3)用透射电子显微镜评估后生动物细胞超微结构的状态，以确定细胞器(如核、高尔基体、氢小体)的状态以及DHAB后生动物是否有特殊的细胞结构。更广泛的影响：无论结果如何，都将获得重要信息。如果后生动物不生活在DHAB中，那么范式转变就没有必要，生理学教科书也不需要重写。如果后生动物生活在DHAB中，那么就需要进行范式转变。由于这项提议是以目前资助的一个研究DHAB原生生物的项目为基础的，大多数拟议的公共宣传努力将与该项目协调。此外，这个短短(两年)的项目将为本科生提供学习分子方法的机会。来自通常无法参加海洋探险的高等学府的学生将被录取。此外，在邮轮期间，世界卫生组织公共网页将提供一个博客。结果将在中学和高中班级以及科学会议上传播。

Intellectual Merit: Although it has been known for many decades that metazoans inhabit anoxic habitats either on a periodic, transient, or semi permanent basis, none have been shown to complete an entire life cycle without access to oxygen. The remarkable recent observation that loriciferan metazoans complete a full life cycle without access to dissolved oxygen raises questions in the fields of physiology and evolution. The habitat from which the anaerobic animals were collected is sediment from a Deep Hypersaline Anoxic Brine (DHAB) in the eastern Mediterranean Sea at a water depth 3km. DHABs are one of the most extreme marine environments known to science, with a water chemistry considered anathema to eukaryotic life. While the possibility of anaerobic metazoa is exciting, there are other potential explanations that warrant investigation before biology textbooks are rewritten. One alternative scenario is that remnant metazoa bodies were inhabited by anaerobic bacteria and/or archaea. The overall goal of this project is to determine if the dominant loriciferan and nematode taxon in each of three DHABs represent living populations. Because remnant DNA can be preserved in anoxic settings for long periods of time, the project will include in situ preservation for RNA analysis. Further, because there is also some chance of RNA preservation in these anoxic sedimentary environments, the study will include analyses of the more ephemeral mRNA and also Transmission Electron Microscopy (TEM). On three ship days added to a funded cruise to sample DHABs for other purposes, an ROV will be used to preserve samples in situ. The specific aims are to: (1) Use RNA and DNA analysis to establish if metazoan ribosomal RNA and functional genes were active at the time of in situ preservation in the dominant two metazoan taxa from each DHAB. (2) Identify the prokaryotes associated with DHAB metazoans using RNA analysis and FISH/CARD FISH. (3) Assess the state of cellular ultrastructure in metazoans using TEM to determine the state of organelles (e.g., nuclei, Golgi, hydrogenosomes) and if DHAB metazoans have specialized cellular structures. Broader Impacts: Regardless of results, significant information will be obtained. If the metazoans are not living in the DHABs, then a paradigm shift is unnecessary and physiology text books do not need to be rewritten. If the metazoans are living in the DHAB, then a paradigm shift is required. Since this proposal piggybacks on a currently funded project to study DHAB protists, most proposed public outreach efforts will be coordinated with that project. Additionally, this short (2 year) project will provide an opportunity for an undergraduate student to learn molecular methods. A student from an institution of higher learning that does not normally have access to oceanographic expeditions will be recruited. Also, during the cruise, a blog will be featured by the WHOI public web page. Results will be disseminated in secondary and high school classes as well as at scientific meetings.