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.

Trace element profiles of the sea anemone Anemonia viridis living nearby a natural CO2 vent

Ocean acidification (OA) is not an isolated threat, but acts in concert with other impacts on ecosystems and species. Coastal marine invertebrates will have to face the synergistic interactions of OA with other global and local stressors. One local factor, common in coastal environments, is trace element contamination. CO2 vent sites are extensively studied in the context of OA and are often considered analogous to the oceans in the next few decades. The CO2 vent found at Levante Bay (Vulcano, NE Sicily, Italy) also releases high concentrations of trace elements to its surrounding seawater, and is therefore a unique site to examine the effects of long-term exposure of nearby organisms to high pCO2 and trace element enrichment in situ. The sea anemone Anemonia viridis is prevalent next to the Vulcano vent and does not show signs of trace element poisoning/stress. The aim of our study was to compare A. viridis trace element profiles and compartmentalization between high pCO2 and control environments. Rather than examining whole anemone tissue, we analyzed two different body compartments-the pedal disc and the tentacles, and also examined the distribution of trace elements in the tentacles between the animal and the symbiotic algae. We found dramatic changes in trace element tissue concentrations between the high pCO2/high trace element and control sites, with strong accumulation of iron, lead, copper and cobalt, but decreased concentrations of cadmium, zinc and arsenic proximate to the vent. The pedal disc contained substantially more trace elements than the anemone's tentacles, suggesting the pedal disc may serve as a detoxification/storage site for excess trace elements. Within the tentacles, the various trace elements displayed different partitioning patterns between animal tissue and algal symbionts. At both sites iron was found primarily in the algae, whereas cadmium, zinc and arsenic were primarily found in the animal tissue. Our data suggests that A. viridis regulates its internal trace element concentrations by compartmentalization and excretion and that these features contribute to its resilience and potential success at the trace element-rich high pCO2 vent.

Ages, lithologie descriptions and mineral analyses from different DSDP holes in the Indian Ocean

The present work is based on mineralogical studies of sand and silt layers from a number of Deep Sea Drilling Project sites in the Indian Ocean belonging to different physiographic provinces of different ages. The minerals can be grouped into two major associations: a hornblende-opaque association with varying amounts of pyroxene, garnet, epidote, zircon, etc. and a biotite-chlorite-muscovite assemblage. The dominance of unstable minerals indicates a first generation, though evidence of reworking is reflected in the zircon and tourmaline grains at some sites. A large variety of minerals at some sites indicates a complex source. The mineral composition is nearly homogeneous at different sites for the entire length of the core, indicating that they have been derived from the same source during the deposition of that interval. However, the provenance changed by tectonic activity, the effect of which has been reflected in the mineralogy of some sites. An attempt was made to describe the mineralogic characteristics and their tectonic interpretations in the Pliocene and Miocene periods in the Ganges and Indus fan sites and also in the Wharton and Mozambique basin sites. Similar attempts could not be made for other ages in other physiographic provinces as the numbers of samples were too few. Within the limited scope, some idea about the mineralogical character of different basins and different physiographic provinces can be obtained from the present study. Mineralogical evidence also suggests very long transport of sediments in the deep sea. Regional variation of mineralogy has resulted due to source, sea-floor configuration, selective removal, reworking by different agencies and the processes operating in the ocean. There is no relation between a particular age and a set mineral assemblage for the Cenozoic sediments of the Indian Ocean.

Pollen and spores in bottom sediments of the Mediterranean Sea

The paper presents results of palynological analysis of deposits from core VITYAZ4779 (length 6.5 m) that was collected from depth 3090 m in the eastern Mediterranean Sea. These palynological data reveal four distinct strata of sediments, each of which was accumulated under different physiographic conditions.

Sedimentology and planktonic foraminiferal dissolution during the latest Cretaceous in the South Atlantic

A high-resolution (10-20 kyr) record of variations in CaCO3 content and dissolution was established for latest Cretaceous (last 0.7 Myr) deep-sea sediments from the South Atlantic Ocean (DSDP Site 516 from the Rio Grande Rise, and sites 525 and 527 from the Walvis Ridge). The degree of fragmentation of planktonic foraminifera (DFP) was used as a measure of calcite dissolution. High negative correlations between DFP and other independent measures of carbonate dissolution (percentage of sand fraction, absolute abundance of planktonic foraminifera, and planktonic/benthic foraminiferal ratio) validate its use as a sensitive index of calcite dissolution in upper Maastrichtian deep-sea sediments. Very high DFP and a significant negative correlation between DFP and CaCO 3 content suggest that Site 516 was located below the foraminiferal lysocline during the entire interval studied. Such a shallow position of the lysocline (paleodepth of Site 516 was 1.2 km) may be explained by "upwelling" of corrosive deep waters along the southern margin of the Rio Grande Rise. Sites 525 and 527 were located above the foraminiferal lysocline; however, three short periods of enhanced dissolution were recognised at Site 525 (paleodepth 1 km) and one interval of strong dissolution was identified at Site 527 (paleodepth 2.7 km). The lack of correspondence between the dissolution regimes at sites from the Walvis Ridge suggests limited deep-water communication across this physiographic barrier. Two of the dissolution maxima recognised at Site 525 correspond to carbonate maxima at Site 527. Variations in "upwelling" intensity along the Walvis Ridge, resulting in fluctuations in primary productivity in this area, may be the proximal cause of both carbonate cycles at Site 527 and dissolution cycles at Site 525. We suggest that development of the bottom Ekman layer between a hypothetical westward geostrophic current and the topographical height of the Rio Grande Rise-Walvis Ridge system may be a plausible hydrodynamical explanation for the proposed "'upwelling" along the southern margin of this topographical structure.