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. 2015 Dec 10:13:105.
doi: 10.1186/s12915-015-0213-6.

Metazoans of redoxcline sediments in Mediterranean deep-sea hypersaline anoxic basins

Joan M Bernhard  1 , Colin R Morrison  2 , Ellen Pape  3 , David J Beaudoin  4 , M Antonio Todaro  5 , Maria G Pachiadaki  6 , Konstantinos Ar Kormas  7 , Virginia P Edgcomb  8
Affiliations

Metazoans of redoxcline sediments in Mediterranean deep-sea hypersaline anoxic basins

Joan M Bernhard et al. BMC Biol. .

Abstract

Background: The deep-sea hypersaline anoxic basins (DHABs) of the Mediterranean (water depth ~3500 m) are some of the most extreme oceanic habitats known. Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB haloclines and brines, loriciferans are the only metazoan reported to inhabit the anoxic DHAB brines. Our goal was to further investigate metazoan communities in DHAB haloclines and brines.

Results: We report observations from sediments of three DHAB (Urania, Discovery, L'Atalante) haloclines, comparing these to observations from sediments underlying normoxic waters of typical Mediterranean salinity. Due to technical difficulties, sampling of the brines was not possible. Morphotype analysis indicates nematodes are the most abundant taxon; crustaceans, loriciferans and bryozoans were also noted. Among nematodes, Daptonema was the most abundant genus; three morphotypes were noted with a degree of endemicity. The majority of rRNA sequences were from planktonic taxa, suggesting that at least some individual metazoans were preserved and inactive. Nematode abundance data, in some cases determined from direct counts of sediments incubated in situ with CellTracker(TM) Green, was patchy but generally indicates the highest abundances in either normoxic control samples or in upper halocline samples; nematodes were absent or very rare in lower halocline samples. Ultrastructural analysis indicates the nematodes in L'Atalante normoxic control sediments were fit, while specimens from L'Atalante upper halocline were healthy or had only recently died and those from the lower halocline had no identifiable organelles. Loriciferans, which were only rarely encountered, were found in both normoxic control samples as well as in Discovery and L'Atalante haloclines. It is not clear how a metazoan taxon could remain viable under this wide range of conditions.

Conclusions: We document a community of living nematodes in normoxic, normal saline deep-sea Mediterranean sediments and in the upper halocline portions of the DHABs. Occurrences of nematodes in mid-halocline and lower halocline samples did not provide compelling evidence of a living community in those zones. The possibility of a viable metazoan community in brines of DHABs is not supported by our data at this time.

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Figures

Fig. 1
Fig. 1
Underwater photographs of representative deep hypersaline anoxic basin halocline interfaces. a Discovery showing floating garbage (aluminum beverage can and plastic item) atop brine. b Portion of L��Atalante halocline showing embayment. c Urania showing emplaced injector cores (607-608cC and 607-608cD on left; E and F on right). Note the impaired visibility of cores on the right due to the brine (B) murkiness. Cores C and D are in the normoxic control/halocline transition (red arrow). d L’Atalante halocline with emplaced injector cores (611cC and 611cF in the upper halocline (UH) zone; 611cA and 611cB in the mid-halocline (MH)). LH, Lower halocline; N, Normoxic normal saline. Outer diameter of pushcorer is 6.9 cm
Fig. 2
Fig. 2
DAPI-labeled copepod crustaceans viewed with epifluorescence microscopy and DAPI optics (377-nm excitation; 447-nm emission). a Molted exoskeleton of a fifth-stage harpacticoid copepodite (L’Atalante lower halocline, 611c14). b Intact fifth-stage harpacticoid copepodite (Discovery lower halocline, 609c14). c Mid- to late-stage cyclopoid copepodite (Discovery lower halocline, 610c9). Note lack of strong DAPI signal in all but appendages of (c). Scales: a, c = 100 μm; b = 200 μm
Fig. 3
Fig. 3
Loriciferan Spinoloricus cinziae, from L’Atalante imaged with differential interference contrast (DIC) (a, c) and DAPI (b, d). a, b Specimen from normoxic control sediments (611c3). Mouth cone (white arrow), lorica spines (black arrow), and scalids (black arrowhead) are visible in (a). Further images are shown in Additional file 1. c, d Specimen from L’Atalante lower halocline sediments (611c14). Mouth cone (white arrow) and scalids (black arrowhead) are visible in (c). Further images are shown in Additional file 2. Scales: a–d = 50 μm
Fig. 4
Fig. 4
Spinoloricus cinziae, details of the lorica. a–c Photos at different focal planes refer to specimen shown in Fig. 3a, b. Arrows indicate the extra spines of the lorica. Scales: a–c = 30 μm
Fig. 5
Fig. 5
Loriciferans. a, b Rugiloricus sp., imaged with differential interference contrast (DIC), from L’Atalante lower halocline sediments (611c17). a Overview showing mouth cone (white arrow) and clavoscalid (cs). b Higher magnification view showing putative oocyte (black arrow). Further images are shown in Additional file 3. c, d Pliciloricus sp., imaged with DIC, from L’Atalante lower halocline sediments (611c17). c Overview showing scalids (black arrowhead), clavoscalids (cs), and mouth cone (white arrow). d Higher magnification of the anterior region (introvert). Further images are shown in Additional file 4. Scales: a, c = 50 μm; b = 30 μm; d = 25 μm
Fig. 6
Fig. 6
Bryozoan. Ctenostomata sp. from Urania normoxic, normal saline/halocline transition (607-608cC). a, b Bright field images showing same individual at different magnifications. c Differential interference contrast image showing internal anatomy of a different specimen. t, Tentacles; g, Gut. Scales: a = 400 μm; b, c = 200 μm
Fig. 7
Fig. 7
Relative abundances (%) of nematode genera in sediment samples, as determined by morphotype analyses. L’Atalante (L’Atl); Discovery (Disc). Parenthetic values represent dive and core number. N, Normoxic, normal saline control; UH, Upper halocline; LH, Lower halocline; MH, Mid-halocline
Fig. 8
Fig. 8
Examples of micrographs of Daptonema nematodes obtained from sediment samples. a–c Morphotype 1 (L’Atalante normoxic normal saline, 611c3). a General habitus of Morphotype 1. b Section showing head and one amphid (arrow) of Morphotype 1. c Spicule of Morphotype 1. d–f Morphotype 2 (Discovery mid-halocline, 610c16). d General habitus of Morphotype 2. e Section showing head of Morphotype 2 and the two amphids (arrows). f Spicule of Morphotype 2. g–i Morphotype 3 (L’Atalante Upper Halocline, 611c5). g General habitus of Morphotype 3. h Section showing head and one amphid (arrow) of Morphotype 3. i Spicule of Morphotype 3. Scales: a, d, g = 100 μm; b, c, h, i = 20 μm; e, f = 10 μm
Fig. 9
Fig. 9
L’Atalante upper halocline nematodes labeled in situ with CellTracker Green (611cC). a–c Epifluorescence micrographs showing labeled nematodes in sediments (480-nm excitation; 520-nm emission). d Paired image of (c), in transmitted light. e–h Epifluorescence images of DAPI staining. e Same nematode shown in (c, d). f–h Nematodes showing clear DAPI staining of nuclei but no evidence of uniform endo- or ectobionts. Scales: a, c–e, g = 250 μm; b = 500 μm; f = 100 μm; h = 50 μm
Fig. 10
Fig. 10
Transmission electron micrographs showing longitudinal sections of three nematodes from L’Atalante normoxia control sediments (611c3). a–c Portions of gut with ingested bacteria (arrowheads). Inset in c: Mitochondria in same specimen. d Nuclei and muscle cells. e Portion showing cells, potentially oocytes, with copious lipid. f Muscle cells and mitochondria. c, Cuticle; *, Mitochondria; mu, Muscle; n, Nuclei; l, Lipids. a–c, e = specimen 1; d = specimen 2; f = specimen 3. Scales: a = 4 μm; b, e = 2 μm; c, d, f = 1 μm; inset = 500 nm
Fig. 11
Fig. 11
Transmission electron micrographs showing longitudinal sections of nematode from L’Atalante upper halocline (611c5). a Overview. Inset: Higher magnification overview, posterior of mouth. b Image of anterior end showing buccal cavity (b) and procorpus (p). c Mitochondria (*), muscle tissues (mu) and cuticle (c). d Cuticle, muscle tissue and nuclei (n) with condensed cromatin. Scales a = 10 μm; b–d = 1 μm; inset = 500 nm
Fig. 12
Fig. 12
Transmission electron micrographs showing longitudinal sections of suspected nematode remnants from L’Atalante lower halocline (611c17). a Specimen 1 showing suspected nematode cuticle and degraded muscle tissue. b Specimen 2 showing remnants of suspected gut and cuticle. c Specimen 2 showing possible anterior end. Note well preserved prokaryote (arrowhead) in lower left. Scales: a, b = 10 μm; c = 2 μm
Fig. 13
Fig. 13
Living Ctenostomata sp. (Bryozoa) (Urania normoxic, normal saline/halocline transition; 607-608cC). a Two specimens labeled with CellTracker Green demonstrating esterase activity. b–d Transmission electron micrographs showing intact organelles such as nuclei (n), mitochondria (m), cilia (c), and ciliary root (cr). Scales: a = 400 μm; b = 2 μm; c, d = 1 μm
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