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

智力优势：尽管几十年来人们都知道后生动物周期性地、短暂地或半永久地居住在缺氧的栖息地，但没有一种动物在没有氧气的情况下完成整个生命周期。 最近观察到的一个值得注意的现象是，兜甲后生动物在没有溶解氧的情况下完成了整个生命周期，这在生理学和进化领域提出了问题。 厌氧动物的栖息地是从地中海东部水深3公里处的深高盐无菌盐水（DHAB）中收集的沉积物。 DHAB是科学界已知的最极端的海洋环境之一，其水化学被认为是真核生物的诅咒。 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调查。 另一种情况是，残留的后生动物体被厌氧细菌和/或古生菌居住。 该项目的总体目标是确定三个DHAB中的每一个中占主导地位的兜虫和线虫分类群是否代表活种群。 由于残余DNA可以在缺氧环境中保存很长一段时间，该项目将包括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RNA保存的机会，该研究将包括对更短暂的mRNA和透射电子显微镜（TEM）的分析。在为其他目的对DHAB进行取样的资助巡航中增加的三个航行日，将使用一个保护器在原地保存样本。 具体目标是：（1）利用RNA和DNA分析来确定在原位保存时，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和功能基因是否活跃。(2)使用RNA分析和FISH/CARD FISH鉴定与DHAB后生动物相关的原核生物。(3)使用TEM评估后生动物细胞超微结构的状态，以确定细胞器的状态（例如，细胞核，高尔基体，氢化酶体），如果DHAB后生动物具有专门的细胞结构。更广泛的影响：无论结果如何，都将获得重要信息。 如果后生动物没有生活在DHAB中，那么范式转变是不必要的，生理学教科书也不需要重写。如果后生动物生活在DHAB中，那么需要范式转换。 由于这项建议是在目前资助的一个研究DHAB原生生物的项目上提出的，因此大多数拟议的公共宣传工作将与该项目协调进行。此外，这个短（2年）项目将提供一个机会，为本科生学习分子方法。 将从通常没有机会参加海洋学考察的高等院校招收一名学生。 此外，在巡航期间，WHOI公共网页将介绍一个博客。 研究结果将在中学和高中课堂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.