The First Metazoa Living in Permanently Anoxic Conditions

Danovaroet al. BMC Biology 2010,8:30 http://www.biomedcentral.eom/1741-7007/8/30 BMC Biology RESEARCH ARTICLE Open Access The first metazoa living in permanently anoxic conditions Roberto Danovaro*1, Antonio Dell'Anno1, Antonio Pusceddu1, Cristina G am bi1, Iben Heiner2 and Reinhardt Mobjerg Kristensen 2 A bstract Background:Several unicellular organisms (prokaryotes and protozoa) can live under permanently anoxic conditions. Although a few metazoans can survive temporarily in the absence of oxygen, it is believed that multi-cellular organisms cannot spend their entire life cycle without free oxygen. Deep seas include some of the most extreme ecosystems on Earth, such as the deep hypersaline anoxic basins of the Mediterranean Sea. These are permanently anoxic systems inhabited by a huge and partly unexplored microbial biodiversity. R esults:During the last ten years three oceanographic expeditions were conducted to search for the presence of living fauna in the sediments of the deep anoxic hypersaline L'Atalante basin (Mediterranean Sea). We report here that the sediments of the L'Atalante basin are inhabited by three species of the animal phylum Loricifera(Spinoloricus nov. sp., Rugiloricus nov. sp. andPliciloricus nov. sp.) new to science. Using radioactive tracers, biochemical analyses, guantitative X-ray microanalysis and infrared spectroscopy, scanning and transmission electron microscopy observations on ultra-sections, we provide evidence that these organisms are metabolically active and show specific adaptations to the extreme conditions of the deep basin, such as the lack of mitochondria, and a large number of hydrogenosome-like organelles, associated with endosymbiotic prokaryotes. Conclusions:This is the first evidence of a metazoan life cycle that is spent entirely in permanently anoxic sediments. Our findings allow us also to conclude that these metazoans live under anoxic conditions through an obligate anaerobic metabolism that is similar to that demonstrated so far only for unicellular eukaryotes. The discovery of these life forms opens new perspectives for the study of metazoan life in habitats lacking molecular oxygen. B ackground in the surface centimetre) [4], These environments are More than 90% of the ocean biosphere is deep (average inhospitable to most marine species [5], except host depth, 3,850 m) and most of this remains unexplored [1], prokaryotes, protozoa and some metazoans that can tol The oceans host life at all depths and across the widest erate these environmental conditions [4,6]. Permanently ranges of environmental conditions (that is, temperature, anoxic conditions in the oceans are present in the subsur salinity, oxygen, pressure), and they represent a huge res face seafloor [7], and among other areas, in the interior of ervoir of undiscovered biodiversity [2,3]. Deep-sea eco the Black Sea (at depths >200 m) [8] and in the deep systems also contain the largest hypoxic and anoxic hypersaline anoxic basins (DHABs) of the Mediterranean regions of the Biosphere. The oxygen minimum zones Sea [9,10]. All of these extreme environments are (OMZ) are widely distributed across all of the oceans, at assumed to be exclusively inhabited by viruses [11], Bac depths generally from 200 m to 1,500 m, and cover teria and Archaea [7-10]. The presence of unicellular approximately 1,150,000 km2. These are characterised by eukaryotes (for example, protozoan ciliates) in anoxic very low oxygen availability (02 < 0.5 mM) and high sul marine systems has been documented for decades [12] phide concentrations in the bottom sediments (>0.1 mMand recent findings have indicated that some benthic for aminifera can be highly adapted to life without oxygen * Correspondence:[email protected] r, [13]. For limited periods of time, a few metazoan taxa can 1 Department of Marine Science, Faculty of Science, Polytechnic University of tolerate anoxic conditions [6,14]. However, so far, there is Marche, Via Brecce Bianche, 60131 Ancona, Italy no proof of the presence of living metazoans that can © 2010 Danovaro et al; licensee BioMed Central Ltd. This ¡san Open Access article distributed under the terms of the Creative Commons B io MedCentral Attribution License (http://creativecommons.0 rg/licenses/by/ 2 .O), which permits unrestricted use, distribution, and reproduction in O any medium, provided the original work is properly cited. Danovaroet al. BMC Biology 2010,8:30 Page 2 of 10 http://www.biomedcentral.eom/1741-7007/8/30 spend their entire life cycle under permanently anoxicto the genera Spinoloricus (Figure le, similar to the new conditions [12]. species of Spinoloricus turbatio, which was recently dis Metazoan meiofauna (multi-cellular organisms of size covered in the deep-sea hydrothermal vents of the Galá ranging from a few micrometres to 1 mm) [15] represent pagos Spreading Centre) [21],Rugiloricus (belonging to 60% of the metazoa abundance on Earth, and have a long the cauliculus-group; Figure le) and Pliciloricus (Figure evolutionary history and high phyletic diversity. They If) [22], include 22 of the 35 animal phyla, six of which are exclu The permanent reducing conditions of anoxic sedi sive of the meiofauna (Gnathostomulida, Micrognatho- ments can preserve dead organisms and their protein for zoa, Gastrotricha, Tardigrada, Kinorhyncha, and a long time, so that microscopic analyses do not provide Loricifera, the most recently described animal phylum) proof of the viability of an organism. However, the abun [16]. These phyla lack larval dispersal in the water col dance of these loriciferans was the highest reported so far umn and spend their entire life cycle in the sediment. Allworld-wide per unit of surface sediment investigated of these characteristics make meiofauna the ideal organ (range: 75 to 701 individuals n r 2). This findingper is se ism for investigating metazoan life in systems without surprising, as only two individuals of the phylum Loric oxygen [17,18]. ifera have been found in the deep Mediterranean Sea over The six DHABs of the Mediterranean Sea are extreme the last 40 years [23-25]. Deep-sea oxygenated sediments environments at depths >3,000 m that have been created in the neighboring of the LAtalante basin were also inves by the flooding of ancient evaporites from the Miocene tigated at the time of sampling as well as in several other period (5.5 million years before the present) [19]. Among occasions since 1989, and we never found one single indi these, the LAtalante basin displays a 30 to 60 m thick vidual of the phylum Loricifera in the entire Ionian basin. hypersaline brine layer with a density of 1.23 g cm '3 [9], Moreover, the analysis of the oxygenated deep-sea sedi which represents a physical barrier that hampers oxygen ments surrounding the LAtalante basin revealed the exchange between the anoxic sediments and the sur dominance of nematodes and copepods (>95% of the rounding seawaters. This basin is therefore completely total meiofaunal abundance; Additional file 3) and the oxygen free, rich in hydrogen sulphide, and hosts anabsence of loriciferans. The density of the Loricifera incredibly diverse and metabolically active prokaryotic extracted from the sediment of the LAtalante basin assemblages that have adapted to these conditions [9]. In (determined by density gradient) was 1.15 to 1.18 g cm '3, 1998, 2005 and 2008 we carried out three oceanographic whereas the density of the brines above the sediment is expeditions to search for the presence of living fauna in significantly higher (1.23 g cm'3). Moreover, the presence the sediments of the anoxic LAtalante basin (Additionalof laminated sediment layers along with the lack of tur- file 1). bidites in the LAtalante basin [26] indicates the lack of lateral transport from adjacent systems. These indepen Results and Discussion dent evidences make very unlikely the sedimentation or In all of the sediments collected from the inner part of the transfer of Loricifera or their carcasses from the oxygen anoxic basin, we found specimens belonging to three aniated sediments surrounding the anoxic basin. mal Phyla: Nematoda, Arthropoda (only Copepoda) and Specimens of the undescribed species of both genera Loricifera. The presence of metazoan meiofauna under Spinoloricus andRugiloricus had a large oocyte in their permanently anoxic conditions has been reported previovary, which showed a nucleus containing a nucleolus ously also from the deep-sea sediments of the Black Sea, (Figure Id, e). This is the first evidence of Loricifera although these records were interpreted as the result of a reproducing in the entire deep Mediterranean basin. rain of cadavers that sunk to the anoxic zone from adja Microscopic analyses also revealed the presence of empty cent oxygenated areas [20]. Our specimens collected exuviae from moulting loriciferans (Figure lg), suggesting from the LAtalante basin were initially stained with a pro that these metazoans did grow in this system. Moreover, tein-binding stain (Rose Bengal) and examined under thescanning electron microscopy confirmed the perfect microscope; here, all of the copepods were empty exu integrity of these loriciferans (Figure 2), while all of the viae, and the nematodes were only weakly stained (sug other meiofaunal taxa were largely damaged or degraded. gesting that they had been dead for a while, Figure la, b), A second expedition was dedicated to the demonstra whereas all of the loriciferans, if stained, were intensely tion of the viability of these loriciferans of the LAtalante coloured (Figure le, d). Differences in the colour intensity basin, through independent experimental approaches. All between live and dead metazoans were confirmed byof the experiments were conducted on deck (101,325 Pa), additional experiments on deep-sea nematodes and copeunder anoxic conditions (in a N2 atmosphere), in the dark pods (Additional file 2). The taxonomic analysis revealed and at thein-situ temperature (ca 14°C) immediately after that the loriciferans collected in the anoxic sediments sample retrieval. In the first investigations, intact and belong to three species that are new to science and belong Danovaroet al.

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