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

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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.