INSIGHTS ON LOCALIZED ROS FORMATION AND PH IN ISOLATED BIVALVE GILLS: A LIFE-IMAGING APPROACH

GEORGINA RIVERA-INGRAHAM; IARA ROCCHETTA; ULF BICKMEYER; DORIS ABELE

Congreso; 106th Annual Meeting of the German Zoological Society.; 2013

Life-imaging techniques (LIT), which consist here in applying specific dyes and quantifying the resulting fluorescence signals under a confocal microscope, have traditionally been used on isolated cells and tissues to study various physiological parameters in-vitro. In the present study we report the successful application of LIT on complete living bivalve gills for the analysis of its physiology and characterization of reactive oxygen species (ROS) formation. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) were mainly concentrated in the interlamellar space of the filaments, while in the outermost areas, associated with the cilia and coinciding with the highest mitochondrial densities (MTK Deep Red 633 staining), showed high and stable concentrations of O2.- (DHE). These peripheral areas were additionally showing the most acidic pH values (as observed with ageladine-a) and mitochondria with low membrane potentials (JC-1), suggesting that the high amounts of ATP required for ciliary beating may be produced via a non-mitochondrial pathway such as the enzymatic activity of an ATP-regenerating kinase. On the ventral bend of the gills, a high number of subepithelial mucus glands (SMG) were observed, which were especially numerous in those areas closer to the mouth. SMG showed to accumulate higher concentrations of O2?- than in the rest of the tissues, while no significant formation of the other ROS analyzed was observed. Given the antimicrobial properties of O2.-, we hypothesize that the release of O2.- into the mucus may be playing an important role in avoiding the development of microbial biofilms on the gills surface.