Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

Phosphorus components and barite in untreated and in carbonate-free sediments of ODP Hole 199-1221C

We determined changes in equatorial Pacific phosphorus (µmol P/g) and barite (BaSO4; wt%) concentrations at high resolution (2 cm) across the Paleocene/Eocene (P/E) boundary in sediments from Ocean Drilling Program (ODP) Leg 199 Site 1221 (153.40 to 154.80 meters below seafloor [mbsf]). Oxide-associated, authigenic, and organic P sequentially extracted from bulk sediment were used to distinguish reactive P from detrital P. We separated barite from bulk sediment and compared its morphology with that of modern unaltered biogenic barite to check for diagenesis. On a CaCO3-free basis, reactive P concentrations are relatively constant and high (323 µmol P/g or ~1 wt%). Barite concentrations range from 0.05 to 5.6 wt%, calculated on a CaCO3-free basis, and show significant variability over this time interval. Shipboard measurements of P and Ba in bulk sediments are systematically lower (by ~25%) than shore-based concentrations and likely indicate problems with shipboard standard calibrations. The presence of Mn oxides and the size, crystal morphology, and sulfur isotopes of barite imply deposition in sulfate-rich pore fluids. Relatively constant reactive P, organic C, and biogenic silica concentrations calculated on a CaCO3-free basis indicate generally little variation in organic C, reactive P, and biogenic opal burial across the P/E boundary, whereas variable barite concentrations indicate significant changes in export productivity. Low barite Ba/reactive P ratios before and immediately after the Benthic Extinction Event (BEE) may indicate efficient nutrient burial, and, if nutrient burial and organic C burial are linked, high relative organic C burial that could temporarily drawdown CO2 at this site. This interpretation requires postdepositional oxidation of organic C because organic C to reactive P ratios are low throughout the section. After the BEE, higher barite Ba/reactive P ratios combined with higher barite Ba concentrations may imply that higher export productivity was coupled with unchanged reactive P burial, indicating efficient nutrient and possibly also organic C recycling in the water column. If the nutrient recycling is decoupled from organic C, the high export production could be indicative of drawdown of CO2. However, the observation that organic C burial is not high where barite burial is high may imply that either C sequestration was restricted to the deep ocean and thus occurred only on timescales of the deep ocean mixing or that postdepositional oxidation (burn down) of organic matter affected the sediments. The decoupling of barite and opal may result from low opal preservation or production that is not diatom based.

Free and dissolved gases in the area of the Calypso hydrothermal vent, Bay of Plenty, New Zealand

Gas composition and hydrochemistry of bottom waters of the Bay of Plenty in the hydrothermally active zone of the Pacific island arc are investigated. Methane content in underwater vents is an order of magnitude greater than that in volcanic exhalations on the land. Salinity, pH, total content of CO2, its partial pressure, and silica content also differ. Correlations between gas parameters, hydrochemical parameters, and biological and microbiological parameters are identified.

Carbonat-free sediment components of ODP Leg 115 holes

From the equatorial Indian Ocean, carbonate-free portions of sediment samples of Paleocene to Miocene calcareous oozes and chalks from Sites 707, 709, and 711 were studied using X-ray diffraction measurements and the scanning electron microscope. Downhole variations in biogenic opal, quartz, barite, and clinoptilolite were investigated. The abundance patterns of these major mineral phases show several similarities and may be used for additional lithologic correlations. Variations in biogenic opal contents reflect biogenic silica productivity. Beside the general pattern, a succession in biogenic silica decrease through time is generally recorded since the Oligocene. This succession started earliest at northernmost Site 711 and latest at southernmost Site 707, including Site 709 within these two. Opal-A variations as well as the barite distribution may be influenced by the paleoposition of the sites in relation to the high-productivity zone, which today lies south of the equator. Authigenic clinoptilolite apparently formed in two different modes. In deeper sediment intervals, clinoptilolite was the last mineral phase formed associated with enhanced silica diagenesis. In late Oligocene to middle Miocene sediments, clinoptilolite was the only authigenic silica phase encountered where otherwise strong opal dissolution was observed. The sponge spicules showed special dissolution features probably related to microbiological activity. Silica concretions mainly composed of opal-CT and authigenic quartz occur in carbonate-rich environments and are formed during later diagenesis when burial depth causes the sediments to reach higher temperatures. Opal-CT concretions in carbonate-free siliceous oozes were found at Site 711 and are probably formed during an early stage of silica diagenesis.

Maps and acoustic profiles of free gas distribution, Western Baltic Sea and Kattgat-Skagerrak area

A shallow gas depth-contour map covering the Skagerrak-western Baltic Sea region has been constructed using a relatively dense grid of existing shallow seismic lines. The digital map is stored as an ESRI shape file in order to facilitate comparison with other data from the region. Free gas usually occurs in mud and sandy mud but is observed only when sediment thickness exceeds a certain threshold value, depending on the water depth of the area in question. Gassy sediments exist at all water depths from approx. 20 m in the coastal waters of the Kattegat to 360 m in the Skagerrak. In spite of the large difference in water depths, the depth of free gas below seabed varies only little within the region, indicating a relatively fast movement of methane in the gas phase towards the seabed compared to the rate of diffusion of dissolved methane. Seeps of old microbial methane occur in the northern Kattegat where a relatively thin cover of sandy sediments exists over shallow, glacially deformed Pleistocene marine sediments. Previous estimates of total methane escape from the area may be correct but the extrapolation of local methane seepage rate data to much larger areas on the continental shelf is probably not justified. Preliminary data on porewater chemistry were compared with the free gas depth contours in the Aarhus Bay area, which occasionally suffers from oxygen deficiency, in order to examine if acoustic gas mapping may be used for monitoring the condition of the bay.