Biometric assessment of deep-sea vent megabenthic communities using multi-resolution 3D image reconstructions

This paper describes a method to survey the distribution of megabenthos over multi-hectare regions of the seafloor. Quantitative biomass estimates are made by combining high-resolution 3D image reconstructions, used to model spatial relationships between representative taxa, with lower-resolution reconstructions taken over a wider area in which the distribution of larger predatory animals can be observed. The method is applied to a region of the Iheya North field that was the target of scientific drilling during the IODP Expedition 331 in 2010. An area of 2.5 ha was surveyed 3 years and 4 months after the site was drilled. More than 100,000 organisms from 6 taxa were identified. The visible effects of drilling on the distribution of megabenthos were confined to a 20 m radius of the artificially created hydrothermal discharges, with the associated densities of biomass lower than observed in nearby naturally discharging areas

Discovery of New Hydrothermal Activity and Chemosynthetic Fauna on the Central Indian Ridge at 18°–20°S

Indian Ocean hydrothermal vents are believed to represent a novel biogeographic province, and are host to many novel genera and families of animals, potentially indigenous to Indian Ocean hydrothermal systems. In particular, since its discovery in 2001, much attention has been paid to a so-called ‘scaly-foot’ gastropod because of its unique iron-sulfide-coated dermal sclerites and the chemosynthetic symbioses in its various tissues. Despite increasing interest in the faunal assemblages at Indian Ocean hydrothermal vents, only two hydrothermal vent fields have been investigated in the Indian Ocean. Here we report two newly discovered hydrothermal vent fields, the Dodo and Solitaire fields, which are located in the Central Indian Ridge (CIR) segments 16 and 15, respectively. Chemosynthetic faunal communities at the Dodo field are emaciated in size and composition. In contrast, at the Solitaire field, we observed faunal communities that potentially contained almost all genera found at CIR hydrothermal environments to date, and even identified previously unreported taxa. Moreover, a new morphotype of ‘scaly-foot’ gastropod has been found at the Solitaire field. The newly discovered ‘scaly-foot’ gastropod has similar morphological and anatomical features to the previously reported type that inhabits the Kairei field, and both types of ‘scaly-foot’ gastropods genetically belong to the same species according to analyses of their COI gene and nuclear SSU rRNA gene sequences. However, the new morphotype completely lacks an iron-sulfide coating on the sclerites, which had been believed to be a novel feature restricted to ‘scaly-foot’ gastropods. Our new findings at the two newly discovered hydrothermal vent sites provide important insights into the biodiversity and biogeography of vent-endemic ecosystems in the Indian Ocean

Identification of Indole Derivatives as Self-Growth Inhibitors of Symbiobacterium thermophilum, a Unique Bacterium Whose Growth Depends on Coculture with a Bacillus sp.▿

Symbiobacterium thermophilum is a syntrophic bacterium whose growth depends on coculture with a Bacillus sp. Recently, we discovered that CO2 generated by Bacillus is the major inducer for the growth of S. thermophilum; however, the evidence suggested that an additional element is required for its full growth. Here, we studied the self-growth-inhibitory substances produced by S. thermophilum. We succeeded in purifying two substances from an ether extract of the culture supernatant of S. thermophilum by multiple steps of reverse-phase chromatography. Electron ionization mass spectrometry and nuclear magnetic resonance analyses of the purified preparation identified the substances as 2,2-bis(3′-indolyl)indoxyl (BII) and 1,1-bis(3′-indolyl)ethane (BIE). The pure growth of S. thermophilum was inhibited by authentic BII and BIE with MICs of 12 and 7 μg/ml, respectively; however, its growth in coculture with Bacillus was not inhibited by BII at the saturation concentration and was inhibited by BIE with an MIC of 14 μg/ml. Both BII and BIE inhibited the growth of other microorganisms. Unexpectedly, the accumulation levels of both BII and BIE in the pure culture of S. thermophilum were far lower than the MICs (<0.1 μg/ml) while a marked amount of BIE (6 to 7 μg/ml) equivalent to the MIC had accumulated in the coculture. An exogenous supply of surfactin alleviated the sensitivities of several BIE-sensitive bacteria against BIE. The results suggest that Bacillus benefits S. thermophilum by detoxifying BII and BIE in the coculture. A similar mechanism may underlie mutualistic relationships between different microorganisms

In situ vital staining for chasing the galatheid crab <i>Shinkaia crosnieri</i> on deep-sea floor

Shinkaia crosnieri, a galatheid crab, has ectosymbiotic bacteria on its ventral setae, and forms very dense crowds in hydrothermally active regions and seep areas. They feed on the symbiotic bacteria and do not chase other animals for predation. To study how they move and behave in jostling crowds, we developed a vital staining to mark their individuals and trace them by using a camera on a remotely operated vehicle (ROV). Among the various dyes examined, Coomasie Brilliant Blue R250 (CBB) stained the galatheid crab the darkest, and its color lasted for more than 5 months in the laboratory at 4–5°C. The ventral setae were strongly stained, while the dorsal shell was weakly stained. The stained galatheid crab survived for more than 8 months. For the in situ staining of S. crosnieri at the Iheya North hydrothermal field in the Okinawa Trough, Japan, we applied a dye solution mixture (20 L) containing CBB and Acid Blue 161 to the galatheid crab population through a funnel equipped on the ROV Hyper-Dolphin. After staining for approximately 5 minutes, more than 18 individuals of S. crosnieri were dyed blue. They were disturbed by the staining process but seemed to be unharmed. The dyed galatheid crabs were identified by the ROV one and two days post staining. They seemed to remain at the place where they were stained.The present vital-staining marking method may present a new way to analyze the behavior and changing habitable range of deep-sea animals like S. crosnieri, and may give us a deeper insight into how these animals behave in a very dense population and explore newer habitats

Cell-specific incorporation of ¹³C-labeled bicarbonate by the <i>Sulfurovum</i> epibionts on <i>S. crosnieri</i> seta in the presence and the absence of thiosulfate according to FISH–Nano-SIMS.

<p>The percent enrichment was estimated using the average ¹³C/¹²C ratio of bulk organic carbon of the epibiotic microbial community before the experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046282#pone-0046282-t002" target="_blank">Table 2</a>.</p>*<p>The filamentous cells were identified as <i>Sulfurovum</i> members by FISH.</p>†<p>The filamentous cells were not identified as the <i>Sulfurovum</i> members by FISH.</p

Time course of the consumption of reduced sulfur compounds by epibiotic microbial community associated with <i>S. crosnieri setae</i>.

<p>The concentration of hydrogen sulfide was examined at the indicated intervals in the absence (black square) and presence (black circle) of the mixed setae samples (A). The unbroken line indicates linear regression analysis for the absence of the mixed setae (<i>y = </i>−2.236<i>x</i>+75.9, <i>R²</i> = 0.89) and the solid line indicates linear regression analysis for the presence of the mixed setae (<i>y = </i>−8.602<i>x</i>+69.4, <i>R²</i> = 0.99) (A). The concentration of thiosulfate was determined at indicated intervals in the absence (black square) and presence (black circle) of mixed setae samples (B). The unbroken line indicates linear regression analysis for the absence of the mixed setae (<i>y = </i>0.079<i>x</i>+75.0, <i>R²</i> = 0.11) and the solid line indicates linear regression analysis for the presence of the mixed setae (<i>y = </i>−4.264<i>x</i>+76.0, <i>R²</i> = 0.99) (B). Statistically significant differences in consumption in the presence and absence of the setae samples at individual time points were determined by unpaired t-test (A, P = 2×10⁻⁷, B, P = 4×10⁻¹⁰).</p

Analyses of epibiont cells after ¹³C bicarbonate tracer experiments performed in the presence of potential thioautotrophic substrates.

<p>Microscopic image of DAPI-stained epibiont cells in a specimen (A). Microscopic image of epibiont cells specifically bound to the EPI653 probe, which indicates that members of the genus <i>Sulfurovum</i> belonging to the class <i>Epsilonproteobacteria</i> were present in the same specimen (B). Scanning electron micrograph of epibiont cells in the same specimen (C). ¹²C-mapping image of epibiont cells in the same specimen performed using Nano-SIMS (D). ¹³C-mapping image of epibiont cells in the same specimen performed using Nano-SIMS (E). The values (secondary ion counts) corresponding to each of the colors are shown in the scale to the right of each map (D and E). The estimated ¹³C/¹²C ratios of the cells that are enclosed by the white lines are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046282#pone-0046282-t003" target="_blank">Table 3</a>. The scale bar is 20 µm.</p