Award: OCE-1061391

Deep-sea Hypersaline Anoxic Basins (DHABs) are some of the most extreme known oceanic habitats. Brines of Mediterranean DHABs occur at ~3.5km water depth. Because DHABs lack dissolved oxygen and are nearly saturated with salt, most believe that eukaryotes cannot inhabit DHABs. Diverse bacterial and protistan communities reportedly inhabit some Mediterranean DHAB haloclines and brines, but only one metazoan type reportedly inhabits the anoxic brines. Our overall goal was to determine if the metazoans in each of three Mediterranean DHABs represent living populations. The proposed specific aims were to: (1) Use RNA and DNA analysis to establish if metazoan ribosomal RNA and selected functional genes were active at the time of in situ preservation in dominant metazoan taxa. (2) Determine if the dominant metazoans showed evidence of prokaryotic symbionts and, if so, to identify those. (3) Assess the state of cellular ultrastructure in metazoans using Transmission Electron Microscopy to determine the state of organelles and if DHAB metazoans have specialized cellular structures. Studies of metazoans in DHAB sediments are rare; existing studies are controversial because reported biological activities could have resulted from prokaryotic contaminants or scavengers. We obtained carefully located samples to test our hypothesis that metazoans living in the hypersaline, low oxygen DHAB sediments were not viable. On a cruise in 2011, using the Remotely Operated Vehicle (ROV) Jason, we collected cores from normal saline, well-oxygenated (control) sediments in close proximity to DHABs, and from where the halocline impinges the seafloor of 3 different DHABs (Urania, Discovery, LÆAtalante). Cores from different zones within DHAB haloclines allowed comparisons of their metazoans. We applied various types of microscopy (reflected light, epifluorescence, Transmission Electron) as well as ribosomal RNA gene iTAG sequencing to determine diversity of prokaryotes and eukaryotes in the sediments. For metazoa, we performed morphotype analyses, abundance calculations, and viability assessments. We also searched for putative symbionts. Nematodes were the most abundant metazoan. We also observed crustaceans, a few loriciferans and some bryozoans. Daptonema was the most abundant nematode; three morphotypes were noted to have a degree of endemicity reflecting habitat chemistry differences. Most rRNA sequences were from planktonic taxa, suggesting preservation of ribosomal RNA from inactive or dead taxa under DHAB conditions. Generally, the highest abundances of nematodes were observed in either the normoxic control samples or in upper halocline samples; nematodes were very rare or absent in lower halocline samples. Nematode ultrastructural analysis indicates the normoxic control specimens were fit, while upper halocline specimens varied with some appearing healthy and others recently dead. Lower halocline specimens had no identifiable organelles (i.e., were dead at the time of collection). The metazoan taxon previously reported living in sediments below the LÆAtalante brine, Loricifera, were only rarely encountered in our samples. Loricifera occurred in both normoxic control samples as well as in Discovery and LÆAtalante haloclines. Loricifera did not appear to have intact tissue, but insufficient numbers were obtained to examine their ultrastructure. It is not clear how any given metazoan taxon could remain viable under both normoxic normal saline and hypersaline anoxic settings. One morphotype of ctenostome (non-calcified) bryozoan was abundant in the seawater/brine transition at the top of the Urania halocline. It is possible this is a new species; a bryozoan expert is examining more specimens. To answer the question posed in our project title: there is evidence for living metazoans in normoxic, normal saline deep-sea Mediterranean sediments and to a lesser degree, in the upper halocline portions of the DHABs studied, but little com...