Benthic Protozoa and Metazoa Living in Deep Anoxic and Hydrogen Sulfide Conditions of the Black Sea: Direct Observations of Actively Moving Ciliophora and Nematoda

Nelli G. Sergeeva<sup>1</sup>; Sofia A. Mazlumyan<sup>1</sup>; Anna Lichtschlag<sup>2</sup>; Moritz Holtappels<sup>2</sup>

Benthic Protozoa and Metazoa Living in Deep Anoxic and Hydrogen Sulfide Conditions of the Black Sea: Direct Observations of Actively Moving Ciliophora and Nematoda

Nelli G. Sergeeva¹

, Sofia A. Mazlumyan¹

, Anna Lichtschlag²

, Moritz Holtappels²

1. Institute of the Biology of the Southern Seas, Sevastopol, Crimea, Russia
2. Max Planck Institute for Marine Microbiology, Germany

Author

Correspondence author
International Journal of Marine Science, 2014, Vol. 4, No. 49 doi: 10.5376/ijms.2014.04.0049
Received: 04 Jun., 2014 Accepted: 02 Jul., 2014 Published: 07 Aug., 2014

This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Preferred citation for this article:

Sergeeva et al., 2014, Benthic Protozoa and Metazoa Living under Anoxic and Sulfide Conditions in the Black Sea: Direct Observations of Actively Moving Ciliophora and Nematoda, International Journal of Marine Science, Vol.4, No.49 1-11 (doi: 10.5376/ijms.2014.04.0049)

Abstract

This study was conducted to search for living fauna in seafloor sediments from nearly 300 m water depth in the outflow area of the Bosporus into the Black Sea. During our investigations we identified seven higher level meiobenthic taxa in the anoxic and sulfidic sediments at 296 m water depth: Gromiida, Ciliophora, Foraminifera (soft-shelled), Nematoda, Kinorhyncha, Harpacticoida and one taxonomically unknown form. Hence, these Black Sea sediments seem to be a natural habitat for some eukaryotes. Furthermore, direct microscopic observations of alive and actively moving fauna demonstrates the presence of two species of ciliates, one species of free-living nematodes, and one unknown organism. As evidence, the activity of these organisms was recorded with a video camera. This indicates that metazoa and protozoa can live in the Black Sea under these more extreme conditions. However, the environmental factors and the specific physiological and biochemical processes used by the benthic fauna to retain metabolic activity and facilitate survival under anoxic and sulfidic conditions are still unknown. As well the distribution of benthos as a whole and of the individual taxa in other sulfidic sediments of the Black Sea needs to be investigated. Nevertheless, the presented observation of alive benthic organisms opens new perspectives to study of metazoan and protozoan life in Black Sea sediments lacking oxygen and containing sulfide.

Keywords

Live eukaryotes; Deep-water; Anoxia; Abundance trends; Black Sea

The Black Sea is a deep marginal meromictic sea and one of the largest and best-studied permanently anoxic water bodies in the world. With increasing water depth dissolved oxygen concentrations decrease, oxygen deficiency (hypoxia) and depletion (anoxia) occurs and hydrogen sulfide starts to build up in the water column below about 200 m. At these depths the Black Sea is often referred to as an environment that is populated only by microbes but remains azoic or lifeless with regard to higher forms of life (Sorokin, 1962; Zhizhchenko, 1974, Kiseleva, 1979). One of the most characteristic features of such euxinic environments is probably the absence of macroscopic benthic fauna. In the absence of bottom-dwelling organisms, responsible for bioturbation and bioirrigation, sediments can accumulate without vertical or horizontal biological mixing (Treude, 2012).

The knowledge of presence of oxygen only in trace amounts limited hydrobiological investigations of the deeper parts of the Black Sea and eukaryotic life under sulfidic conditions is still under debate. As result, it is still argued if eukaryotic life in the sulfidic and anoxic Black sea waters and sediments can exists (Zaitsev et al., 2007, 2008). However, the first direct evidence of living multicellular animals under anoxic and sulfidic conditions was provided by the findings of alive and metabolically active meiofauna in the deep hypersaline L'Atalante basin (Mediterranean Sea) at water depths of more than 3,000 m, where the anoxic sediments were colonized by a natural population of loriciferans that were able to reproduce (Danovaro et al., 2010). Adaptations to this permanently anoxic condition with sulfide concentrations of up to 2.9 mmol/L imply that these organisms have developed specific survival mechanisms. Currently, it is unknown what type of metabolism may drive the growth of Loricifera in this inhospitable environment, however the presence of hydrogenosome-like organelles and associated prokaryote-like rods suggests that the process may be similar to that known from several types of anaerobic protozoa (Oren, 2012).

Benthic protozoans and metazoans have been studied in the Black Sea for more than 30 years in the oxic/suboxic/anoxic transition zones as well as in the permanently sulfidic and anoxic zones of the Black Sea (Kolesnikova et al., 2014; Sergeeva, 2001, 2003a, b; Sergeeva and Gulin, 2009; Sergeeva and Dovdal, 2014; Sergeeva and Zaika, 2008; Zaika, 2008; Zaitsev et al., 1987). Meiofauna have been found in the bottom sediments under permanently sulfidic conditions (depth range 400~2250 m) (Sergeeva, 2001, 2003c). Later, additional data has been obtained concerning taxonomical richness and abundance of deep-water protozoa and metazoa in sediments underlying hypoxic and anoxic water column (Sergeeva et al., 2012, 2013). This revealed that certain ciliate and foraminifera taxa can occur in anoxic sediments and can live in the absence of oxygen in the Black Sea (e.g. Sergeeva et al., 2012).

Systematic studies of fauna from the deeper parts of the Black Sea, using sediments collected with a TV-guided multicorer (TV-MUC), began in 1994 and 2006-2010 (MEGASEEPS, HERMES and HYPOX EU projects), where the area of the deep-water Dnieper Canyon and the outlet area of the Istanbul Strait’s (Bosporus) of the Black Sea was studied in more detail. Three oceanographic expeditions were conducted to survey fauna in oxic/suboxic/anoxic and permanently sulfidic sediments: RV ‘Meteor’ cruise 72/2 (February–March 2007); the RV ‘Arar’ cruise (November 2009), RV ‘Maria S. Merian’ cruise 15/1 (April–May 2010) (Sergeeva et al., 2012, 2013). Near the Dnieper Canyon, discovered Nematoda (120~240 m) and Harpacticoida (120~170 m) population included specimens of all sizes and life stages, such as gravid females containing eggs. In the sediments harpacticoid Archesola typhlops (Sars, 1920) was found, which population included adult females, males, and copepodites at different stages (Kolesnikova, 2010). As well the nematode fauna was unique and included 90 species and 9 genera unknown from the Black Sea and 19 species, 9 genera and 1 family recognized for the first time in this basin. The fauna included stenobiontic and eurybiontic forms adapted to live in the redox zone (Sergeeva et al., 2012).

During a study of bottom sediments associated with methane gas hydrates in the Sorokin Trough (NE Black Sea), for the first time an alive actively moving endemic species of Cladocera was discovered in the anoxic and sulfidic area at depths of 1990 and 2140 m. Cladocera was described as Pseudopenilia bathyalis, the type species of a new genus (Sergeeva 2003c, 2004 a) and family Pseudopenilidae (Korovchinsky & Sergeeva, 2008).

1 Study area

In the Black Sea the combined effect of great depth (> 2000 m), restricted inflow of saline waters and large river water input, creates a basin-wide water-column stratification and a chemocline separating an oxic zone above 100 to 150 m water depth from an anoxic and sulfidic zone below. A suboxic zone exists between the oxygenized and anoxic waters. In the suboxic layer all oxygen is depleted and no sulfide can be found in the water column. This layer is important for biogeochemical and redox reactions (Codispoti et al., 1991; Murray et al., 1989, Kuypers et al., 2003). In the Bosporus outflow area of the Black Sea oxygenated Mediterranean water with high density is injected into the anoxic layers of the stratified Black Sea water column. In the Black Sea basin oxygen is normally absent below 150 m water depth, however in the Bosporus outflow area, oxygen (>2 µmol/L) was detected in warm saline water intrusions as deep as 230 m (Friedrich et al., 2013).

2 Methods

2.1 Fauna sampling

Bottom fauna was studied in the Bosporus outlet area of the Black Sea during the RV ‘Maria S. Merian’ cruise MSM 15/1 in April 2010 (Figure 1). This study presents meio- and macrobenthos analyzes from 8 stations sampled along a downslope transect from oxygenated bottom water (97.2 m) to anoxic bottom water (295.7 m) (Figure 1, Table 1). Sediments were sampled with a TV-guided multicorer (TV-MUC) and with a gravity corer (GC. ITU Corer) and intact sediment stratification was assured by visual inspection. Sediments were sectioned in 0–1, 1–2, 2–3 and 3–4 and 4–5cm intervals and combined results from these upper 5cm are presented. At each station the density of benthos was calculated from the number of animals found per sediment interval of a TV-MUC core (9.5 cm diameter or 70.8 cm²) or an ITU core (7 cm diameter or 38.5 cm²) and extrapolated to m² of seafloor area. Sediment sections were preserved in 75 % alcohol, which is known to conserve morphological structures of fauna without distortion. To avoid damaging the calcareous taxa we did no prior fixation in formalin. Sediment samples were washed with distilled water through sieves with mesh sizes of 1 mm and 63 μm. The fraction that retained in the sieves was stained with Rose Bengal solution, a dye traditionally used to separate living and dead or decaying organisms (Bernhard, 2000; Bernhard et al., 2006; Danovaro et al., 2010; Grego et al., 2013) and sorted in water under a binocular microscope for ‘live’ (stained) organisms. We extracted only those specimens that stained intensely with Rose Bengal and that showed no signs of morphological damage. All isolated macro- and meiobenthos was counted and identified to higher taxa level. All protozoa and metazoa fauna larger than 63 μm were categorized as meiobenthos.

Figure 1 Benthic stations in the Istanbul Strait's (Bosporus) outlet area of the Black Sea (April, 2010)

Table 1 Water depths and coordinates of meiobenthos stations in the Bosporus outlet area of the Black Sea (‘Maria S. Merian’ cruise 15/1. 2010)

To reveal the presence of living fauna in the deeper and potentially anoxic sediments of the Bosporus outflow area, unfixed sediment samples from 296 m water depth were studied. The sediment (Station 262) was transferred to a Petri dish (90 mm diameter) immediately after sampling and analyzed visually using light microscopy. These observations of living benthic fauna were conducted in bottom sediment without subjecting it to mechanical and physical effects i.e. washing through sieves or use of thermal and electrical appliances. Active movements of some organisms were filmed with a video camera (Canon DIGITAL IXUS 90 IS Camera).

2.2 Geochemical analyzes

Dissolved oxygen, salinity and temperature of the water column were measured with a conductivity-temperature-depth (CTD) probe, equipped with an oxygen sensor (SeaBird Electronics). The oxygen concentration was calibrated against Winkler titration (Winkler 1888). A total of 59 CTD casts were performed in the Bosporus outlet area between 12 and 18 April 2010. Of these, 10 CTD casts were performed to map the oxygen distribution along a 4 km long transect crossing the sediment sampling stations. Oxygen concentrations were measured from the surface down to 5 m above the sea floor. Oxygen and sulfide concentrations were measured in water samples collected with Niskin bottles attached to the CTD rosette.

At the same sites (Figure 1) where sediment cores for benthos analyzes were collected, pore water was extracted from sediments with Rhizons (type: CSS, Rhizosphere Research Products), fixed with ZnAc for total sulfide concentration analyzes and analyzed according to the method of Cline (1969).

3 Results

3.1 Oxygen and sulfide concentrations in the water column

Oxygen and sulfide concentrations measured in water samples from the CTD rosette are plotted as a function of water density (Figure 2). The disappearance of oxygen and the onset of sulfide were found at a density of approximately 1016 kg/m³. Oxygen concentrations in the water column along the sediment stations are shown in Figure 3. Further offshore, at water depths between 150~300 m, the oxicline was situated at 120 m depth and was characterized by steep oxygen gradients. The onset of sulfide as marked by the 1016 kg/m³ density isoline was between 150 and 200 m depth. Further inshore, at water depths less than 150 m, the oxicline intersects with the seafloor causing oxygen gradients to become less steep. The bottom water at the sediments Station 285 (100 m depth, not shown in Figure 3) was fully oxygenated, whereas the oxygen concentrations were decreased to ~10 µmol/L at Station 243 (154 m). At Station 224 (200 m) and 333 (200 m) the bottom water was anoxic but not yet sulfidic, whereas at all other stations (203, 204, 262, 263, 332; > 250 m) the bottom water was sulfidic.

Figure 2 Compiled oxygen and sulfide concentrations in the water sampled by the CTD rosette as a function of density. The onset of the sulfidic layer is at 1016 kg m^-3

Figure 3 Oxygen concentrations in the water column along the sediment sampling stations. Station numbers denote the stations where sediment was sampled. The white lines represent density isolines. The 1016 kg m^-3 isoline marks the onset of sulfide

3.2 Sulfide concentrations in pore water

Sulfide concentrations in the sediment pore waters at the different stations are displayed in Figure 4. In the upper 30 cm of sediments from 100 m (Station 285) and 150 m (Station 243) water depth no sulfide was detected in the pore waters. At 200 m (Station 224,333) water depth sulfide was occasionally presents in the pore waters of the sediment. Sulfide concentrations were variable here, however, in the two sampled stations no free sulfide was found in the upper 5 cm of the sediments. Only at 250 m (Station 250 m) water depth sulfide was also detected close to the sediment surface.

Figure 4 Dissolved sulfide concentrations in pore waters along the depth transect at the Bosporus outlet area of the Black Sea

4 Microscopic observations of animals

Using direct microscopic observations to test viability of benthos, we found two ciliates species, one species of free-living nematodes and one unknown organism, alive and actively moving in sediments of Station 262 at 296 m water depth (Table 1). These observations are further described in 4.1~4.3 (Figure 7, Figure 8; Figure 10, Figure 11). This visual observation of living organisms demonstrates that metazoa and protozoa can inhabit Black Sea sediments in areas that were previously thought to be devoid of higher life. The indigenous eukaryotic fauna observed active in the fresh, not sieved sediment samples were recorded on video (Supplementary material). As these sediments were rather liquid black fine sediment and had a strong smell of the hydrogen sulfide and the overlying water column was anoxic and sulfidic during our sampling (Figure 2, Figure 3), the presence of these indigenous organisms in the sediments provides evidence that some eukaryotes can live under anoxic/sulfidic conditions in the Black Sea at 296 m water depth.

Analyses of the sediment samples yield a characteristic fauna of benthic organisms and we have identified six to seven higher level taxa: Gromiida, Ciliophora, Foraminifera (soft-shelled), Nematoda, Kinorhyncha, Harpacticoida and one taxonomically unknown form in two cores from Stations 296 m.

In the muddy sediments at a 252 m water depth (Station 203 and 204), we found a large number of the macrobenthic oligochaetes Tubificoides sp. (body length 5~7 mm). Presence of these macrobenthic oligochaetes in high density and actively moving will lead to a strong bioturbation of the sediment on other studied habitats (Figure 5).

Figure 5 Tubificoides sp (Oligochaeta) in the seafloor sediments of Station 224 and Station 263

In the same sediment sample three macrobenthic polychaetes species Aricidea sp. and Capitellidae g. sp. and Vigtorniella zaikai were identified (Figure 6).

Figure 6 Aricidea sp. (A) and Vigtorniella zaikai (B) in the seafloor sediments of Station 224-1 and Station 263: A – B – general view of individual polychaetes. C - D – possible endosymbiontes in different parts of the body of Aricides. E- endosymbiontes in the body of Vigtorniella

4.1 Microscopic observations of alive Protozoa

Observations with the microscope showed active movement and changes in the body shape of both Ciliophora species detected in the sediments (Figures 7 and 8). The two different morphotypes of ciliates showed different motion activity. Ciliophora gen. sp. 1 changed its location in the Petri dish, slowly rotating around its axis, keeping the form of the body (Figure 7. A-E). In contrast, Ciliophora gen. sp. 2 was actively moving along the bottom of the Petri dish inside the silt and detritus particles, changing body length and diameter, but also the shape of the body (Figure 8 A-K). Significant changes in ciliates body shape impeded the process of its identification.

Figure 7 Movements of Ciliophora species, A–E: Ciliophora gen. sp. 1 (Station 262)

Figure 8 Movements of Ciliophora species. A–K: Ciliophora gen. sp. 2 (Station 262)

4.2. Microscopic observations of alive Metazoa

From the sediment of the same station active movement was observed for representative of the free-living Nematoda Theristus sp. (Figures 9 and Figure 10). This species was dominating the nematodes fauna in samples from the anoxic and sulfidic sediments.

Figure 9 Nematoda species Theristus sp. (male) found in the seafloor sediments of Station 224 and Station 263: A– general view; B–head; C–spicula; D– includes (conjectural symbionts) in the body

Figure 10 Movements of Nematoda Theristus sp; (Station 262)

The size of the individuals of the nematode species Theristus sp. was rather large (1.55~1.94 mm). As seen in photos during the observation (> 2 two hours) this nematode changed actively its orientation in the Petri dish, extending its body and coiling (Figure 10). These results confirm that some metazoa organisms can live in the anoxic and sulfidic sediments of the Black Sea.

4.3 Microscopic observations of an alive taxonomically unknown form

One unusual finding was made in the meiobenthos and its systematic affiliation is currently uncertain (Figure 11). We can assume that this is a representative of a single polyp of Anthozoa. Its body was clearly subdivided into three parts: the apical (head), middle (body) and lower part (base), which plays a role for attachment to the substrate. Apparently, in situ this animal lived attached as during microscopy its orientation was vertical to the substrate (in this case the bottom of the Petri dishes). Apical and basal parts have the ability to diminish or widen (expand) their volume and their color was more light compared to the more dark median part.

Figure 11 Movements of taxonomically unknown form (station MS M15/262-1)

5 Taxonomic structure and abundance of benthos

The macrobenthos that was identified in sediments of the Bosporus outflow area between 100 m and 252 m water depth included the following high taxa: Porifera, Coelenterata, Mollusca: (Bivalvia, Gastropoda), Annelida: (Polychaeta, Oligochaeta, Nemertini), Echinodermata, Arthropoda (Crustacea). Highest numbers of individuals were found at 150 m and at 200 m water depth (Figures 12 and Figure 13).

Figure 12 Mean abundance (10^2*ind*m^-2)of macrobenthos found in cores (0~5 cm) sampled in the Bosporus outlet area of the Black Sea

Figure 13 Distribution of bottom fauna (full core sample 0~5 cm) at Bosporus Strait outlet area of the Black Sea

The meiobenthos between 100 m and 296 m included the following 23 high taxa: Ciliophora, Coelenterata, Gromiida, Foraminifera (soft-shelled and hard-shelled forms). Rotifera, Gastrotricha, Turbellaria, Nematoda, Kinorhyncha, Oligochaeta, Polychaeta, Nemertini, Bivalvia, Gastropoda, Ophiuroidea, Harpacticoida, Cumacea, Amphipoda, Tanaidacea, Ostracoda, Acari, Tardigrada, Tunicata and an unknown taxon. At the same depths (117 m~296 m) filamentous fungi were first discovered (Sergeeva and Kopytina, 2014). The meiobenthos taxa made up the main proportion of the fauna in the sediments (Figure 13). However, distribution of benthos abundance was irregular along observed stations at the same depth (252 m) (Figure 14).

Figure 14 Distribution of the benthic fauna from cores sampled in different stations at 250 m water depth (0~5 cm)

6 Discussion

In general, metazoans are obligate aerobes. Certain oligochaetes and nematodes, however, can live parts of their life cycles under anoxic or even sulfidic conditions (e.g. Fenchel and Findlay, 1995; Fenchel, 2012; Bernhard & Sen Gupta, 1999; Koho and Pina-Ochoa, 2012). The same has been shown for other multicellular organisms as well, which are known to be able to live a part of the life cycle without oxygen (Fenchel, 2012). Recently, Danovaro et al. (2010) have demonstrated that also metazoan (lociferans) can be adapted to the permanently anoxic, highly saline and sulfidic conditions in the deep Mediterranean L’Atalante Basin. These meiofaunal organisms are metabolically active and show specific adaptations to survive these extreme conditions. As well some nematode species can inhabit sulfidic environments (Nuß, 1984; Wetzel et al., 2001). The findings of anaerobic multicellular animals in the sediment below the anoxic brine in the depths of the Mediterranean Sea may be not too surprising, as in general, when one searches in unusual environments, one finds unusual organisms (Oren, 2012).

During our study we found traces of bioturbation in hydrogen sulfide containing sediments. This was most likely caused by the high densities and active movements of macrobenthic oligochaetes (Tubificoides sp.), polychaetes (Aricidea sp. and Capilellidaeg. sp.), large nematodes (Theristus sp.) and we observed movements of other fauna (Gromia, Foraminifera, Hydrozoa) in the sediment. This is despite the presence of sulfide in the sediment. Sulfide is generally toxic for most organisms, but some species have developed strategies for detoxification (Nuß, 1984). For example, species of the genus Tubificoides are able to tolerate hypoxia and sulfidic conditions by symbiosis with chemoautotrophic bacteria (Dubilier, 1994).

The overall abundance of benthic fauna from sediments collected in the Bosporus outflow area of the Black Sea suggest that the oxic/anoxic transition zone supports a rich protozoan and metazoan community with high abundances of especially meiobenthos taxa. It is well known that all metazoans have to be in contact with oxygen at least during part of their lifetime (Fenchel, 2012) as they require oxygen for synthetic pathways producing sterols, collagen, and quinone tanning (Barrett, 1991). Our data show presence of fauna also in samples obtained from areas with anoxic bottom water. The high nematodes and harpacticoides densities detected in these anoxic sediment samples suggest that some benthic eukaryotes (protozoa and metazoa) in this area can tolerate anoxic and sulfidic conditions. As well, ciliates, gromiids and foraminiferans were found in the upper 0-5 cm of the anoxic sediments. Altogether, these results confirm our early conclusions that some forms of benthos can possibly adapt to hypoxia/anoxia and sulfide-rich environments (Sergeeva et al., 2013).

It is considered that the permanent reducing conditions of anoxic sediments can preserve dead organisms and their protein for a long time, so that microscopic analyses of fixed bottom sediments do not provide proof of the viability of fauna (Zaitsev et al., 2007, 2008). To proof that benthos is alive, viability must be tested, in particular the metabolic activity (Danovaro et al., 2010) and the ability to reproduce (Kolesnikova and Sergeeva, 2011). Our observations of alive fauna proved the presence of eukaryotic life in the anoxic and sulfidic sediments of the Black Sea. It is known that meiobenthos can be unevenly distributed in sediments (Sergeeva et al. 2012, 2013) and this can explain the different distribution of fauna in cores sampled with a TV MUC at similar depth (Figure 14). This uneven distribution of meiobenthos visible is likely related to variable environmental conditions, such as oxygen concentration, presence of hydrogen sulfide in bottom water and sediment, but also the heterogeneity of habitats and possible differences in the degree of bioturbation. One explanation might be the special characteristic of the Bosporus outflow area, where oxygenated Mediterranean water is injected into the anoxic water of the Black Sea. As a results, the more saline and heavier Mediterranean water sinks rapidly and traces of oxygen can be found occasionally at depths of up to 300 m (Özsoy et al., 2001), so more than 100 m below the zone were oxygen usually is depleted in the Black Sea. However, during our sampling campaign in April 2010 no signs (neither in the oxygen nor in the salinity signal) of an active Bosporus inflow were recorded in the 59 CTD casts that were performed within 7 days. Despite the regular Mediterranean inflow carrying dissolved oxygen into deeper waters and their penetration to considerable depths of the Black Sea, in this study we show that periodic anoxic conditions can prevail, leading to a buildup of sulfide close to the sediment-water interface at 200 m or even sulfide efflux at 250 m (Figure 4).

Presence of some species of ciliates and harpacticoides in sediment containing sulfide is supported by the studies conducted in the last decades. It is known from previous studies, that specific groups of protozoa, fungi and lower metazoa can be found in the sediments of coastal “sulfidic systems”. Populations of the harpacticoides Darcythompsonia fairlensis (T. Scott. 1899) were found in sulfidic patches in different shallow areas of the Black Sea. The population disappeared with sediment oxygenation, which reflects adaptation to the sulfidic environment. D. fairlensis was recognized for the first time in this basin (Kolesnikova and Sergeeva, 2011) and is one of the few species of the phylum Harpacticoida, which has been found to inhabit hypoxic or even anoxic sediments in other water bodies (Kunz, 1961). The discovered harpacticoides population included adult females, males, and copepodites at different stages, ready to breed. These might indicate that even the complete life cycle takes place in these sediments, subject to anoxia and sulfidic conditions.

7 Conclusion

Direct microscopy observations of alive and actively moving ciliates and free-living nematodes provides evidence that sediments underlying a sulfidic water column (200~300 m water depth) can be a natural habitat for eukaryotes (i.e. metazoan: Nematoda, Polychaeta, Kinorhyncha and Harpacticoida and protozoan: Gromiida, Foraminifera (soft-shelled), Ciliophora). These eukaryotic organisms seem to be indigenous habitants of the anoxic/sulfidic sediments. However, the environmental factors and the specific physiological and biochemical processes of the benthic fauna to retain the metabolically activity and facilitate survival are currently still unknown. As well further studies have to be conducted on the distribution of benthos as a whole and its individual taxa in sediments underlying the sulfidic water column in the Black Sea. The presented observations of alive benthic organisms are opening new perspectives for the study of metazoan and protozoan life in Black Sea habitats depleted in oxygen and enriched in sulfide.

Acknowledgements

The study was funded by the Max-Planck-Gesellschaft (www.mpg.de), the European Union project ‘‘HYPOX - In situ monitoring of oxygen depletion in hypoxic ecosystems of coastal and open seas and land-locked water bodies’’, EC grant 226213 (http://ec.europa.eu/research/fp7). We are grateful to Professor Antje Boetius for providing an opportunity to participate in RV ‘Maria S. Merian’ cruise 15/1. The paper benefited from the comments of two anonymous reviewers.

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