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.

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.

Peak areas and heights for minerals of ODP Leg 113-696B (Table 1)

Seismic data acquired over the eastern shelf and margin of the South Orkney microcontinent, Antarctica, have shown a high-amplitude reflection lying at a sub-bottom two-way traveltime (TWT) of 0.5-0.8 s. There appear to be two causes for the reflection which apply in different parts of the shelf. The more widespread cause of the reflection is a break-up unconformity associated with the opening of Jane Basin to the east. This is clearly seen where reflections in the underlying sequence are discordant. In contrast, in Eotvos Basin and the southeastern part of Bouguer Basin, the high-amplitude reflection in places cuts across bedding and is interpreted to be caused by silica diagenesis. A post-cruise analysis of core samples from Site 696 in Eotvos Basin by X-ray diffraction (XRD) and scanning electron microscopy (SEM) revealed the presence of a silica diagenetic front at 520-530 mbsf. The position of the unconformity at this site is uncertain, but probably coincides with a change of detrital input near 548 mbsf. Fluctuations of physical properties related to the depth of the diagenetic front are difficult to separate from those related to the variation of detrital composition over the same depth interval. Correlation of the drilling record with the seismic record is difficult but with a synthetic seismogram it is demonstrated that diagenesis is the probable cause of the high-amplitude reflection. In Bouguer Basin at Site 695 the depth of the high-amplitude reflection was not reached by drilling; however, the reflection is probably also caused by silica diagenesis because of the biogenic silica-rich composition of the sediments cored. The estimated temperatures and ages of the sediments at the depths of the high-amplitude reflections at Sites 695 and 696 compare favorably with similar data from other diagenetic fronts of the world. The high-amplitude reflection in Bouguer Basin is commonly of inverse polarity, possibly caused either by interference between reflections from several closely-spaced reflecting layers, such as chert horizons, or by free gas trapped near the diagenetic front.

Mineralogy and chemistry of bentonitic clays from the Wombat and Exmouth Plateaus

Bentonites (i.e., smectite-dominated, altered volcanic ash layers) were recovered in Berriasian to Valanginian hemipelagic sediments of the Wombat Plateau (Site 761) and southern Exmouth Plateau (Site 763). They are compared to coeval bentonites in eupelagic sediments of the adjacent Argo Abyssal Plain (Sites 261 and 765) and Gascoyne Abyssal Plain (Site 766). A volcaniclastic origin with dacitic to rhyolitic ash as parent material is suggested by the abundance of well-ordered montmorillonite, fresh to altered silicic glass shards, volcanogenic minerals (euhedral sanidine, apatite, and long-prismatic zircon), and volcanic rock fragments, and by a vitroclastic ultrafabric (smectitized glass shards). We distinguish (1) pure smectite bentonites with a white, pink, or light gray color, a waxy appearance, and a very homogeneous, cryptocrystalline smectite matrix (water-free composition at Site 761: 68.5% SiO2, 0.27% TiO2, 19.1% Al2O3, 3.3% Fe2O3, 0.4%-1.1% Na2O, and 0.6% K2O) and (2) impure bentonitic claystones containing mixtures of volcanogenic smectite and pyroclastic grains with terrigenous and pelagic components. The ash layers were progressively altered during diagenesis. Silicic glass was first hydrated, then slightly altered (etched with incipient smectite authigenesis), then moderately smectitized (with shard shape still intact), and finally completely homogenized to a pure smectite matrix without obvious relict structures. Euhedral clinoptilolite is the latest pore-filling or glass-replacing mineral, postdating smectite authigenesis. Volcanic activity was associated with continental breakup and rapid subsidence during the "juvenile ocean phase." Potential source areas for a Neocomian post-breakup volcanism include the Wombat Plateau, Joey and Roo rises, Scott Plateau, and Wallaby Plateau/Cape Range Fracture Zone. Westward-directed trade winds transported silicic ash from these volcanic source areas to the Exmouth Plateau into the adjacent abyssal plains. The Wombat Plateau bentonites are interpreted as proximal ash turbidites.

(Table 3, page 280) Chemical composition of micronodules from a series of stations and sediment depths from Areas C, F, G and K in the Pacific Ocean from R/V Sonne Cruise SO06

Compositional data for coexisting manganese nodules, micronodules, sediments and pore waters from five areas in the equatorial and S.W. Pacific have been obtained. This represents the largest study of its type ever undertaken to establish the distribution of elements between the various phases within the sediment column. The composition of manganese nodules, micronodules and sediments (on a carbonate-free basis) shows marked differences between the equatorial high productivity zone and the low productivity region of the S.W. Pacific. In the case of the nodules, th is reflects an increased supply of transition elements (notably Ni, Cu and Zn) to the nodules as a result of the in situ dissolution of siliceous tests within the sediment column in the equatorial Pacific high productivity zone. Micronodules display similar, but somewhat different, compositions to those of the associated nodules in each area. Micronodule composition is therefore influenced by the same basic factors that control nodule composition, but is modified by dissolution of the micronodules in situ within the sediment column. Locally, as in the area immediately south of the Marquesas Fracture Zone, the micronodule population is contaminated by small, angular volcanic rock fragments; this leads to apparently anomalous micronodule compositions. Micronodules appear to be a transient feature in the sediment column, especially in the equatorial Pacific. Dissolution of micronodules in the sediment column therefore represents an important source of elements for the growth of manganese nodules in the equatorial Pacific. Sediment composition is markedly influenced by the carbonate content. On a carbonate-free basis, the sediments from the equatorial high productivity zone are quite distinct in composition from those in the S.W. Pacific. This reflects differences in the lithology of the sediments. In the Aitutaki Passage, the local influence of volcanoclastic material in sediment composition has been established. The major cations and anions in pore waters measured here show no major differences between equatorial and S.W. Pacific sediments. Silica is, however, higher in equatorial Pacific pore waters reflecting the dissolution of siliceous tests in these sediments.