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.

Living benthic foraminiferal assemblages in surface samples of the Arabian Sea

The distribution, biomass, and diversity of living (Rose Bengal stained) deep-sea benthic foraminifera (>30 µm) were investigated with multicorer samples from seven stations in the Arabian Sea during the intermonsoonal periods in March and in September/October, 1995. Water depths of the stations ranged between 1916 and 4425 m. The distribution of benthic foraminifera was compared with dissolved oxygen, % organic carbon, % calcium carbonate, ammonium, % silica, chloroplastic pigment equivalents, sand content, pore water content of the sediment, and organic carbon flux to explain the foraminiferal patterns and depositional environments. A total of six species-communities comprising 178 living species were identified by principal component analysis. The seasonal comparison shows that at the western stations foraminiferal abundance and biomass were higher during the Spring Intermonsoon than during the Fall Intermonsoon. The regional comparison indicates a distinct gradient in abundance, biomass, and diversity from west to east, and for biomass from north to south. Highest values are recorded in the western part of the Arabian Sea, where the influence of coastal and offshore upwelling are responsible for high carbon fluxes. Estimated total biomass of living benthic foraminifera integrated for the upper 5 cm of the sediment ranged between 11 mg Corg m**-2 at the southern station and 420 mg Corg m**-2 at the western station. Foraminifera in the size range from 30 to 125 ?m, the so-called microforaminifera, contributed between 20 and 65% to the abundance, but only 3% to 28% to the biomass of the fauna. Highest values were found in the central and southern Arabian Sea, indicating their importance in oligotrophic deep-sea areas. The overall abundance of benthic foraminifera is positively correlated with oxygen content and pore volume, and partly with carbon content and chloroplastic pigment equivalents of the sediment. The distributional patterns of the communities seem to be controlled by sand fraction, dissolved oxygen, calcium carbonate and organic carbon content of the sediment, but the critical variables are of different significance for each community.

Abundance of living coccolithophores in the northern Arabian Sea (Table 2)

The present study was conducted to provide information about living coccolithophores from the northern Arabian Sea as potential proxies in palaeoceanographic studies. In all, 71 plankton samples from 16 stations collected in September 1993 were analysed for their contents of living coccolithophores. Absolute abundances range from less than 400 coccospheres per litre in surface waters to 35 000 spheres per litre at intermediate water depths. From 49 identified taxa, nine species contribute significant cell numbers of more than 2000 coccospheres per litre and comprise more than 10% of the communities in at least one sample. Important species are (in approximate order of cell abundances): Gephyrocapsa oceanica, Florisphaera profunda, Oolithotus antillarum, Calciosolenia murrayi, Umbellosphaera irregularis, Emiliania huxleyi, Umbellosphaera tenuis, Calciopappus rigidus, and Algirosphaera robusta. At most profiles, a vertical succession of coccolithophore species was found. Calciosolenia murrayi and C. rigidus were restricted to surface waters, whereas high numbers of F. profunda and A. robusta occurred at depths below 40 m. The coccolithophore communities reflected the local oceanographic situation and seemed to be more dependent on mixed layer depth and nutrient availability than on temperature and salinity changes. Additionally, synecologic competition with diatoms in part controlled the species composition and generally reduced the abundance of coccolithophores. Synecological and ecological tolerances of species were discussed with the help of cluster analysis.

Living benthic foraminifers from the central Arctic Ocean

Fifty short sediment cores collected with a multiple corer and five box cores from the central Arctic Ocean were analysed to study the ecology and distribution of benthic foraminifers. To work out living faunal associations, standing stock and diversity, separate analyses of living (Rose Bengal stained) and dead foraminifers were carried out for the sediment surface. The size fractions between 63 and 125 µm and >125 µm were counted separately to allow comparison with former Arctic studies and with studies from the adjacent Norwegian-Greenland Sea, Barents Sea and the North Atlantic Ocean. Benthic foraminiferal associations are mainly controlled by the availability of food, and competition for food, while water mass characteristics, bottom current activity, substrate composition, and water depth are of minor importance. Off Spitsbergen in seasonally ice-free areas, high primary production rates are reflected by high standing stocks, high diversities, and foraminiferal associations (>125 µm) that are similar to those of the Norwegian-Greenland Sea. Generally, in seasonally ice-free areas standing stock and diversity increase with increasing food supply. In the central Arctic Ocean, the oligotrophic permanently ice-covered areas are dominated by epibenthic species. The limited food availability is reflected by very low standing stocks and low diversities. Most of these foraminiferal associations do not correspond to those of the Norwegian-Greenland Sea. The dominant associations include simple agglutinated species such as Sorosphaerae, Placopsilinellae, Komokiacea and Aschemonellae, as well as small calcareous species such as Stetsonia horvathi and Epistominella arctica. Those of the foraminiferal species that usually thrive under seasonally ice-free conditions in middle bathyal to lower bathyal water depth are found under permanently ice-covered conditions in water depths about 1000 m shallower, if present at all.